Control nucleic acids for multiple parameters

ABSTRACT

The present invention concerns the amplification of at least a first and a second target nucleic acid that may be present in at least one fluid sample using an internal control nucleic acid for qualitative and/or quantitative purposes.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/368,979 filed on Jul. 29, 2010. The presentapplication is a continuation of U.S. Non-provisional application Ser.No. 14/079,155 filed on Nov. 13, 2013, which is a continuation of U.S.Non-provisional application Ser. No. 13/192,317 filed on Jul. 27, 2011(issued as U.S. Pat. No. 8,609,340 on Dec. 17, 2013), which claims thebenefit of U.S. Provisional Application No. 61/368,979 filed on Jul. 29,2010. The entire disclosures of the above-referenced prior applicationsare hereby incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention belongs to the field of in-vitro diagnostics.Within this field, it particularly concerns the amplification of atleast a first and a second target nucleic acid that may be present in atleast one fluid sample using an internal control nucleic acid forqualitative and/or quantitative purposes.

BACKGROUND OF THE INVENTION

In the field of molecular diagnostics, the amplification of nucleicacids from numerous sources has been of considerable significance.Examples for diagnostic applications of nucleic acid amplification anddetection are the detection of viruses such as Human Papilloma Virus(HPV), West Nile Virus (WNV) or the routine screening of blood donationsfor the presence of Human Immunodeficiency Virus (HIV), Hepatitis-B(HBV) and/or C Virus (HCV). Furthermore, said amplification techniquesare suitable for bacterial targets such as mycobacteria, or the analysisof oncology markers.

The most prominent and widely-used amplification technique is PolymeraseChain Reaction (PCR). Other amplification reactions comprise, amongothers, the Ligase Chain Reaction, Polymerase Ligase Chain Reaction,Gap-LCR, Repair Chain Reaction, 3 SR, NASBA, Strand DisplacementAmplification (SDA), Transcription Mediated Amplification (TMA), andQβ-amplification. Automated systems for PCR-based analysis often makeuse of real-time detection of product amplification during the PCRprocess in the same reaction vessel. Key to such methods is the use ofmodified oligonucleotides carrying reporter groups or labels.

It has been shown that amplification and detection of more than onetarget nucleic acid in the same vessel is possible. This method iscommonly termed “multiplex” amplification and requires different labelsfor distinction if real-time detection is performed.

It is mostly desirable or even mandatory in the field of clinicalnucleic acid diagnostics to control the respective amplification usingcontrol nucleic acids with a known sequence, for qualitative(performance control) and/or quantitative (determination of the quantityof a target nucleic using the control as a reference) purposes. Giventhe diversity especially of diagnostic targets, comprising prokaryotic,eukaryotic as well as viral nucleic acids, and given the diversitybetween different types of nucleic acids such as RNA and DNA, controlnucleic acids are usually designed in a specific manner. In brief, thesecontrols usually resemble the target nucleic acid for which they serveas control in order to mimic their properties during the process. Thiscircumstance applies for both qualitative and quantitative assays. Incase multiple parameters are to be detected in a single or in parallelexperiments, usually different controls resembling different targetnucleic acids are employed, such as e.g. in Swanson et al. (J. Clin.Microbiol., (2004), 42, pp. 1863-1868). Stocher et al. (J. Virol. Meth.(2003), 108, pp. 1-8) discloses a control nucleic acid in which multiplevirus-specific competitive controls are comprised on the same DNAmolecule.

The present invention provides a controlled amplification method using adifferent approach that displays various advantages.

SUMMARY OF THE INVENTION

The present invention provides a process for isolating andsimultaneously amplifying at least a first and a second target nucleicacid that may be present in one or more samples, the process comprisingthe steps of:

-   -   a) adding an internal control nucleic acid to each of the one or        more samples;    -   b) combining a solid support material and the one or more        samples in one or more vessels for a period of time, under        conditions sufficient to permit nucleic acids comprising the        target nucleic acids and the internal control nucleic acid to be        immobilized on the solid support material;    -   c) isolating the solid support material from the other material        present in the samples in a separation station;    -   d) purifying the target nucleic acids in the separation station        and washing the solid support material one or more times with a        wash buffer;    -   e) contacting the purified target nucleic acids and the purified        internal control nucleic acid with one or more amplification        reagents, comprising at least one distinct set of primers for        each of the target nucleic acids and for the internal control        nucleic acid in at least two reaction vessels, wherein at least        a first reaction vessel comprises at least the first target        nucleic acid, and at least a second reaction vessel comprises at        least the second target nucleic acid, and wherein the second        target nucleic acid is absent from the first reaction vessel;    -   f) incubating in the reaction vessels the purified target        nucleic acids and the purified internal control nucleic acid        with the one or more amplification reagents for a period of        time, under conditions sufficient for an amplification reaction        indicative of the presence or absence of the target nucleic        acids to occur; and    -   g) detecting and measuring signals generated by the        amplification products of the target nucleic acids and being        proportional to the concentration of the target nucleic acids,        and detecting and measuring a signal generated by the internal        control nucleic acid;        wherein the conditions for amplification and detection in        steps d) to g) are identical for the at least first and second        purified target nucleic acids and the internal control nucleic        acid, and wherein the sequence of the internal control nucleic        acid is identical for the at least first and second purified        target nucleic acids.

The invention further provides wherein the presence of an amplificationproduct of the internal control nucleic acid is indicative of anamplification occurring in the reaction mixture even in the absence ofamplification products for one or more of the target nucleic acids, inan embodiment further comprising the following step:

-   -   h) determining the quantity of one or more of the target nucleic        acids.

The invention further provides a process wherein the amplificationreagents comprise a polymerase with reverse transcriptase activity, theprocess further comprising between step e) and step f) the step ofincubating in the reaction vessels the purified nucleic acids with theone or more amplification reagents for a period of time and underconditions suitable for transcription of RNA by the polymerase withreverse transcriptase activity to occur.

The invention provides an embodiment wherein the internal controlnucleic acid is DNA, wherein the internal control nucleic acid is RNA,and further, wherein the internal control nucleic acid is an armorednucleic acid. Further, the invention provides wherein the sequence ofthe internal control nucleic acid is different from the sequences of theother nucleic acids present in the one or more samples, wherein thesequence of the internal control nucleic acid is derived from anaturally occurring genome, wherein the sequence of the internal controlnucleic acid is scrambled, wherein the internal control nucleic acid hasa melting temperature from 50° C. to 90° C., and wherein the internalcontrol nucleic acid has a length of up to 500 bases. Further providedis the process wherein the sequence of the internal control nucleic acidhas a GC content of 30% to 70%, wherein the internal control nucleicacid has a concentration between LOD and 20×LOD, and wherein theinternal control nucleic acid has a concentration between 20×LOD and5000×LOD. In another embodiment, the more than one internal controlnucleic acid is added in step a), but only one of the internal controlnucleic acids is amplified in step f).

DESCRIPTION OF THE INVENTION

The present invention provides a method for the controlled amplificationof at least a first and a second target nucleic acid that may be presentin a fluid sample.

In a first aspect, the invention relates to a process for isolating andsimultaneously amplifying at least a first and a second target nucleicacid that may be present in one or more fluid samples, said processcomprising the automated steps of:

-   -   a. adding an internal control nucleic acid to each of said fluid        samples    -   b. combining together a solid support material and said one or        more fluid samples in one or more vessels for a period of time        and under conditions sufficient to permit nucleic acids        comprising the target nucleic acids and the internal control        nucleic acid to be immobilized on the solid support material    -   c. isolating the solid support material from the other material        present in the fluid samples in a separation station    -   d. purifying the nucleic acids in said separation station and        washing the solid support material one or more times with a wash        buffer    -   e. contacting the purified target nucleic acids and the purified        internal control nucleic acid with one or more amplification        reagents comprising at least one distinct set of primers for        each of said target nucleic acids and for said internal control        nucleic acid in at least two reaction vessels, wherein at least        a first reaction vessel comprises at least said first target        nucleic acid and at least a second reaction vessel comprises at        least said second target nucleic acid and wherein the second        target nucleic acid is absent from the first reaction vessel    -   f. incubating in said reaction vessel said purified target        nucleic acids and said purified internal control nucleic acid        with said one or more amplification reagents for a period of        time and under conditions sufficient for an amplification        reaction indicative of the presence or absence of said target        nucleic acids to occur    -   g. detecting and measuring signals generated by the        amplification products of said target nucleic acids and being        proportional to the concentration of said target nucleic acids,        and detecting and measuring a signal generated by said internal        control nucleic acid,        wherein the conditions for amplification and detection in        steps d. to g. are identical for said at least first and second        purified target nucleic acids and said internal control nucleic        acid, and wherein the sequence of said internal control nucleic        acid is identical for said at least first and second purified        target nucleic acids.

The present invention allows for the development of simultaneous assayson a plurality of parameters and/or nucleic acid types while using thesame internal control nucleic acid sequence for said differentparameters and/or nucleic acid types. Therefore, it contributes toreducing the overall complexity of the corresponding experiments onvarious levels: For instance, only one internal control nucleic acidsequence has to be designed and added to the respective amplificationmixes, thus saving the time and costs for designing and synthesizing orbuying multiple control nucleic acid sequences. The assay or assays canbe streamlined, and the risk of handling errors is reduced. In addition,the more different control nucleic acid sequences are employed in oneassay or parallel assays carried out simultaneously under the sameconditions, the more complex it may result to adjust the respectiveconditions. Moreover, with a single control suitable for a plurality ofnucleic acids, said control can be dispensed from a single source e.g.into different vessels containing said different target nucleic acids.Within the scope of the invention, the single control nucleic acidsequence may also serve as a qualitative and as a quantitative control.

As a further advantage of the method described above, the testing of aparticular biological sample for other nucleic acids in possiblesubsequent experiments need not involve another sample preparationprocedure with the addition of a different internal control nucleicacid, since the control used in the invention can be used to control theamplification of different nucleic acids. Thus, once an internal controlnucleic acid has been added, other parameters may be tested in the samesample under the same conditions.

The internal control nucleic acid can be competitive, non-competitive orpartially competitive.

A competitive internal control nucleic acid carries essentially the sameprimer binding sites as the target and thus competes for the sameprimers with the target. While this principle allows a good mimicry ofthe respective target nucleic acid due to their similar structure, itcan lower the amplification efficiency with regard to the target nucleicacid or acids and thus lead to a less sensitive assay.

A non-competitive internal control nucleic acid has different primerbinding sites than the target and thus binds to different primers.Advantages of such a setup comprise, among others, the fact that thesingle amplification events of the different nucleic acids in thereaction mixture can take place independently from each other withoutany competition effects. Thus, no adverse effects occur regarding thelimit of detection of the assay as can be the case in a competitivesetup.

Finally, in an amplification using a partially competitive setup therespective control nucleic acid and at least one of the target nucleicacids compete for the same primers, while at least one other targetnucleic acid binds to different primers.

The fact that the method described above involves a distinct set ofprimers for each of said target nucleic acids and for said internalcontrol nucleic acid renders the method considerably flexible. In thisnon-competitive setup it is not necessary to introduce target-specificbinding sites into the control nucleic acid as in the case of acompetitive setup, and the drawbacks of a competitive setup as mentionedabove are avoided. In a non-competitive setup, the internal controlnucleic acid has a sequence different from any target sequences, inorder not to compete for their primers and/or probes. For example, thesequence of the internal control nucleic acid can be different from theother nucleic acid sequences in the fluid sample. As an example, if thefluid sample is derived from a human, the internal control nucleic acidmay not have a sequence which also endogenously occurs within humans.The difference in sequence should thus be at least significant enough tonot allow the binding of primers and/or probes to the respectiveendogenous nucleic acid or acids under stringent conditions and thusrender the setup competitive. In order to avoid such interference, thesequence of the internal control nucleic acid used in the invention canbe derived from a source different from the origin of the fluid sample.For example, it is derived from a naturally occurring genome, forexample a plant genome, or from a grape genome. In an embodiment, anucleic acid derived from a naturally occurring genome is scrambled. Asknown in the art, “scrambling” means introducing base mutations in asequence to a certain extent. For example, the sequence of the internalcontrol nucleic acid used in the invention is substantially altered withrespect to the naturally occurring gene it is derived from.

The process comprising the automated steps mentioned above also displaysvarious additional advantages:

It has been a challenge in the prior art that the number of differenttarget nucleic acids in a multiplex assay carried out in a singlereaction vessel is limited by the number of appropriate labels. In areal-time PCR assay, for example, the potential overlap of fluorochromespectra has a great impact on assay performance (risk of false positiveresults, lower precision etc.) Therefore, the respective fluorophoreshave to be carefully selected and spectrally well separated in order toassure the desired performance of a diagnostic test. Typically, thenumber of different usable fluorophores corresponds to a single-digitnumber of PCR instrument fluorescence channels.

In contrast, in the process described supra, the internally controlledamplification of at least a first and a second target nucleic acid takesplace in at least two different reaction vessels, allowing for thesimultaneous amplification of a higher number of different targetnucleic acids, since signals in different reaction vessels can bedetected independently from each other. Still, within the scope of thepresent invention are embodiments wherein in one or more of the multiplereaction vessels multiplex reactions are performed, thereby multiplyingthe number of targets that may be amplified simultaneously and under thesame conditions. In such embodiments, the internal control nucleic acidserves as a control for the different target nucleic acids within avessel as well as different target nucleic acids in different vessel.

Thus, one aspect of the invention relates to the process describedsupra, wherein at least two target nucleic acids are amplified in thesame reaction vessel.

For example, it may be preferred to amplify the first, but not thesecond target nucleic acid in the first reaction vessel and only thesecond, but not the first target nucleic acid in the second reactionvessel, e.g. depending on the sample and/or the target nucleic acid oracids in question.

Therefore, a further embodiment of the invention is the processdescribed above, wherein the first target nucleic acid is absent fromthe second reaction vessel.

Especially if a fluid sample is suspected to contain target nucleicacids from different organisms, or even the different organisms as such,or if it is not clear which of the different nucleic acids or organismsmay be present in said sample, an advantageous embodiment of theinvention is the process described above, wherein the first targetnucleic acid and the second target nucleic acid are from differentorganisms.

A further aspect of the invention is the process described above,wherein the first and/or the second target nucleic acid is a non-viralnucleic acid.

Also, an aspect of the invention is the process described supra, whereinthe first and/or the second target nucleic acid is a bacterial nucleicacid.

As described before, the method described above is useful forqualitatively or quantitatively controlling the amplification of atleast a first and a second target nucleic acid.

Qualitative detection of a nucleic acid in a biological sample iscrucial e.g. for recognizing an infection of an individual. Thereby, oneimportant requirement for an assay for detection of a microbialinfection is that false-negative or false-positive results be avoided,since such results would almost inevitably lead to severe consequenceswith regard to treatment of the respective patient. Thus, especially inPCR-based methods, a qualitative internal control nucleic acid is addedto the detection mix. Said control is particularly important forconfirming the validity of a test result: At least in the case of anegative result with regard to the respective target nucleic acid, thequalitative internal control reaction has to perform reactive withingiven settings, i.e. the qualitative internal control must be detected,otherwise the test itself is considered to be inoperative. However, in aqualitative setup, said qualitative internal control does notnecessarily have to be detected in case of a positive result. Forqualitative tests, it is especially important that the sensitivity ofthe reaction is guaranteed and therefore strictly controlled As aconsequence, the concentration of the qualitative internal control mustbe relatively low so that even in a situation e.g. of slight inhibitionthe qualitative internal control is not be detected and therefore thetest is invalidated.

Thus, an aspect of the invention is the process described above, whereinthe presence of an amplification product of said internal controlnucleic acid is indicative of an amplification occurring in the reactionmixture even in the absence of amplification products for one or more ofsaid target nucleic acids.

On the other hand and in addition to mere detection of the presence orabsence of a nucleic acid in a sample, it is often important todetermine the quantity of said nucleic acid. As an example, stage andseverity of a viral disease may be assessed on the basis of the viralload. Further, monitoring of any therapy requires information on thequantity of a pathogen present in an individual in order to evaluate thetherapy's success. For a quantitative assay, it is necessary tointroduce a quantitative standard nucleic acid serving as a referencefor determining the absolute quantity of a target nucleic acid.Quantitation can be effectuated either by referencing to an externalcalibration or by implementing an internal quantitative standard.

In the case of an external calibration, standard curves are created inseparate reactions using known amounts of identical or comparablenucleic acids. The absolute quantity of a target nucleic acid issubsequently determined by comparison of the result obtained with theanalyzed sample with said standard function. External calibration,however, has the disadvantage that a possible extraction procedure, itsvaried efficacy, and the possible and often not predictable presence ofagents inhibiting the amplification and/or detection reaction are notreflected in the control.

This circumstance applies to any sample-related effects. Therefore, itmight be the case that a sample is judged as negative due to anunsuccessful extraction procedure or other sample-based factors, whereasthe target nucleic acid to be detected and quantified is actuallypresent in the sample.

For these and other reasons, an internal control nucleic acid added tothe test reaction itself is of advantage. When serving as a quantitativestandard, said internal control nucleic acid has at least the followingtwo functions in a quantitative test:

i) It monitors the validity of the reaction.ii) It serves as reference in titer calculation thus compensating foreffects of inhibition and controlling the preparation and amplificationprocesses to allow a more accurate quantitation.

Therefore, in contrast to the qualitative internal control nucleic acidin a qualitative test which must be positive only in a target-negativereaction, the quantitative control nucleic acid in a quantitative testhas two functions: reaction control and reaction calibration. Thereforeit must be positive and valid both in target-negative andtarget-positive reactions.

It further has to be suited to provide a reliable reference value forthe calculation of high nucleic acid concentrations. Thus, theconcentration of an internal quantitative control nucleic acid needs tobe relatively high.

Therefore, an aspect of the invention is the process described above,further comprising the following step:

-   -   h. determining the quantity of one or more of said target        nucleic acids.

The internally controlled process described above requires considerablyless hands-on time and testing is much simpler to perform than real-timePCR methods used in the prior art. The process offers a major advantagee.g. in the field of clinical virology as it permits parallelamplification of several viruses in parallel experiments. The process isparticularly useful in the management of post-transplant patients, inwhom frequent viral monitoring is required. Thereby said processfacilitates cost-effective diagnosis and contributes to a decrease inthe use of antiviral agents and in viral complications andhospitalizations. This equally applies to the field of clinicalmicrobiology. In general, efficiencies will be gained in fasterturnaround time and improved testing flexibility. Consequently, thisleads to a decrease in the number of tests requested on a patient tomake a diagnosis, and potentially shorter hospital stays (e.g. if adiagnosis can be provided sooner, patients requiring antimicrobialtherapy will receive it sooner and thus recover earlier). In addition,patients show less morbidity and therefore cause fewer costs related tosupportive therapy (e.g., intensive care related to a delay in diagnosisof sepsis). Providing a negative result sooner can have importantimplications for the overprescription of antibiotics. For example, if atest result obtained by the process according to the invention is ableto rule out the pathogen more quickly than with a standard real-time PCRmethod, then the clinician will not be forced to use empiricalantibiotics. Alternatively, if empirical antibiotics are used, theduration of the respective treatment can be shortened.

With respect to designing a specific test based on the process accordingto the invention, the skilled artisan will particularly, but not only,benefit from the following advantages:

-   -   a reduction in software complexity (leading to a reduced risk of        programming errors)    -   focusing of assay development efforts on the chemistry        optimization instead of the chemistry plus the instrument        control parameters    -   much more reliable system since a single process is always used        and the hardware can be optimally designed to perform this        protocol    -   the skilled artisan performing the internally controlled process        described above is provided with the flexibility to run multiple        different assays in parallel as part of the same process    -   cost reduction.

In the sense of the invention, “purification”, “isolation” or“extraction” of nucleic acids relate to the following: Before nucleicacids may be analyzed in a diagnostic assay e.g. by amplification, theytypically have to be purified, isolated or extracted from biologicalsamples containing complex mixtures of different components. Often, forthe first steps, processes are used which allow the enrichment of thenucleic acids. To release the contents of cells or viral particles, theymay be treated with enzymes or with chemicals to dissolve, degrade ordenature the cellular walls or viral particles. This process is commonlyreferred to as lysis. The resulting solution containing such lysedmaterial is referred to as lysate. A problem often encountered duringlysis is that other enzymes degrading the component of interest, e.g.deoxyribonucleases or ribonucleases degrading nucleic acids, come intocontact with the component of interest during the lysis procedure. Thesedegrading enzymes may also be present outside the cells or may have beenspatially separated in different cellular compartments prior to lysis.As the lysis takes place, the component of interest becomes exposed tosaid degrading enzymes. Other components released during this processmay e.g. be endotoxins belonging to the family of lipopolysaccharideswhich are toxic to cells and can cause problems for products intended tobe used in human or animal therapy.

There is a variety of means to tackle the above-mentioned problem. It iscommon to use chaotropic agents such as guanidinium thiocyanate oranionic, cationic, zwitterionic or non-ionic detergents when nucleicacids are intended to be set free. It is also an advantage to useproteases which rapidly degrade the previously described enzymes orunwanted proteins. However, this may produce another problem as saidsubstances or enzymes can interfere with reagents or components insubsequent steps.

Enzymes which can be advantageously used in such lysis or samplepreparation processes mentioned above are enzymes which cleave the amidelinkages in protein substrates and which are classified as proteases, or(interchangeably) peptidases (see Walsh, 1979, Enzymatic ReactionMechanisms. W. H. Freeman and Company, San Francisco, Chapter 3).Proteases used in the prior art comprise alkaline proteases (WO98/04730) or acid proteases (U.S. Pat. No. 5,386,024). A protease whichhas been widely used for sample preparation in the isolation of nucleicacids in the prior art is proteinase K from Tritirachium album (see e.g.Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N. Y., 1989) which isactive around neutral pH and belongs to a family of proteases known tothe person skilled in the art as subtilisins. Especially advantageousfor the use in lysis or sample preparation processes mentioned above isthe enzyme esperase, a robust protease that retains its activity at bothhigh alkalinity and at high temperatures (EP 1 201 753).

In the sample preparation steps following the lysis step, the componentof interest is further enriched. If the non-proteinaceous components ofinterest are e.g. nucleic acids, they are normally extracted from thecomplex lysis mixtures before they are used in a probe-based assay.

There are several methods for the purification of nucleic acids:

-   -   sequence-dependent or biospecific methods as e.g.:        -   affinity chromatography        -   hybridization to immobilized probes    -   sequence-independent or physico-chemical methods as e.g.:        -   liquid-liquid extraction with e.g. phenol-chloroform        -   precipitation with e.g. pure ethanol        -   extraction with filter paper        -   extraction with micelle-forming agents as            cetyl-trimethyl-ammonium-bromide        -   binding to immobilized, intercalating dyes, e.g. acridine            derivatives        -   adsorption to silica gel or diatomic earths        -   adsorption to magnetic glass particles (MGP) or            organo-silane particles under chaotropic conditions

Particularly interesting for purification purposes is the adsorption ofnucleic acids to a glass surface although other surfaces are possible.Many procedures for isolating nucleic acids from their naturalenvironment have been proposed in recent years by the use of theirbinding behavior to glass surfaces. If unmodified nucleic acids are thetarget, a direct binding of the nucleic acids to a material with asilica surface may be preferred because, among other reasons, thenucleic acids do not have to be modified, and even native nucleic acidscan be bound. These processes are described in detail by variousdocuments. In Vogelstein B. et al., Proc. Natl. Acad. USA 76 (1979)615-9, for instance, a procedure for binding nucleic acids from agarosegels in the presence of sodium iodide to ground flint glass is proposed.The purification of plasmid DNA from bacteria on glass dust in thepresence of sodium perchlorate is described in Marko M. A. et al., Anal.Biochem. 121 (1982) 382-387. In DE-A 37 34 442, the isolation ofsingle-stranded M13 phage DNA on glass fiber filters by precipitatingphage particles using acetic acid and lysis of the phage particles withperchlorate is described. The nucleic acids bound to the glass fiberfilters are washed and then eluted with a methanol-containing Tris/EDTAbuffer. A similar procedure for purifying DNA from lambda phages isdescribed in Jakobi R. et al., Anal. Biochem. 175 (1988) 196-201. Theprocedure entails the selective binding of nucleic acids to glasssurfaces in chaotropic salt solutions and separating the nucleic acidsfrom contaminants such as agarose, proteins or cell residue. To separatethe glass particles from the contaminants, the particles may be eithercentrifuged or fluids are drawn through glass fiber filters. This is alimiting step, however, that prevents the procedure from being used toprocess large quantities of samples. The use of magnetic particles toimmobilize nucleic acids after precipitation by adding salt and ethanolis more advantageous and described e.g. in Alderton R. P. et al., S.,Anal. Biochem. 201 (1992) 166-169 and PCT GB 91/00212. In thisprocedure, the nucleic acids are agglutinated along with the magneticparticles. The agglutinate is separated from the original solvent byapplying a magnetic field and performing a wash step. After one washstep, the nucleic acids are dissolved in a Tris buffer. This procedurehas a disadvantage, however, in that the precipitation is not selectivefor nucleic acids. Rather, a variety of solid and dissolved substancesare agglutinated as well. As a result, this procedure can not be used toremove significant quantities of any inhibitors of specific enzymaticreactions that may be present. Magnetic, porous glass is alsocommercially available that contains magnetic particles in a porous,particular glass matrix and is covered with a layer containingstreptavidin. This product can be used to isolate biological materials,e.g., proteins or nucleic acids, if they are modified in a complexpreparation step so that they bind covalently to biotin. Magnetizableparticular adsorbents proved to be very efficient and suitable forautomatic sample preparation. Ferrimagnetic and ferromagnetic as well assuperparamagnetic pigments are used for this purpose. Magnetic glassparticles and methods using them are described in WO 01/37291.Particularly useful for the nucleic acid isolation in the context of theinvention is the method according to R. Boom et al. (J Clin Microbiol.28 (1990), 495-503).

The term “solid support material” comprises any of the solid materialsmentioned above in connection with the immobilization of nucleic acids,e.g. magnetic glass particles, glass fibers, glass fiber filters, filterpaper etc., while the solid support material is not limited to thesematerials.

Thus, an aspect of the invention is the process described above, whereinthe solid support material comprises one or more of the materialsselected from silica, metal, metal oxides, plastic, polymers and nucleicacids. In an embodiment of the invention, the solid support material ismagnetic glass particles.

“Immobilize”, in the context of the invention, means to capture objectssuch as e.g. nucleic acids in a reversible or irreversible manner.Particularly, “immobilized on the solid support material”, means thatthe object or objects are associated with the solid support material forthe purpose of their separation from any surrounding media, and can berecovered e.g. by separation from the solid support material at a laterpoint. In this context, “immobilization” can e.g. comprise theadsorption of nucleic acids to glass or other suitable surfaces of solidmaterials as described supra. Moreover, nucleic acids can be“immobilized” specifically by binding to capture probes, wherein nucleicacids are bound to essentially complementary nucleic acids attached to asolid support by base-pairing. In the latter case, such specificimmobilization leads to the predominant binding of target nucleic acids.

After the purification or isolation of the nucleic acids including thetarget nucleic acids from their natural surroundings, analysis may beperformed e.g. via the simultaneous amplification described supra.

“Simultaneously”, in the sense of the invention, means that two actions,such as amplifying a first and a second or more nucleic acids, areperformed at the same time and under the same physical conditions. Inone embodiment, simultaneous amplification of the at least first andsecond target nucleic acids is performed in one vessel. In anotherembodiment, simultaneous amplification is performed with at least onenucleic acid in one vessel and at least a second nucleic acid in asecond vessel, at the same time and under the same physical conditions,particularly with respect to temperature and incubation time wherein theinternal control nucleic acid mentioned above is present each of saidvessels.

The “first target nucleic acid” and the “second target nucleic acid” aredifferent nucleic acids.

A “fluid sample” is any fluid material that can be subjected to adiagnostic assay targeting nucleic acids and may be derived from abiological source. For example, said fluid sample is derived from ahuman and is a body liquid. In an embodiment of the invention, the fluidsample is human blood, urine, sputum, sweat, swab, pipettable stool, orspinal fluid.

The term “reaction vessel” comprises, but is not limited to, tubes orthe wells of plates such as microwell, deepwell or other types ofmultiwell plates, in which a reaction for the analysis of the fluidsample such as e.g. reverse transcription or a polymerase chain reactiontakes place. The outer limits or walls of such vessels are chemicallyinert such that they do not interfere with the analytical reactiontaking place within. For example, the isolation of the nucleic acids asdescribed above is also carried out in a multiwell plate.

In this context, multiwell plates in analytical systems allow parallelseparation and analyzing or storage of multiple samples. Multiwellplates may be optimized for maximal liquid uptake, or for maximal heattransfer. A multiwell plate for use in the context of the presentinvention can be optimized for incubating or separating an analyte in anautomated analyzer. For example, the multiwell plate is constructed andarranged to contact a magnetic device and/or a heating device.

Said multiwell plate, which is interchangeably termed “processing plate”in the context of the invention, comprises:

-   -   a top surface comprising multiple vessels with openings at the        top arranged in rows. The vessels comprise an upper part, a        center part and a bottom part. The upper part is joined to the        top surface of the multiwell plate and comprises two longer and        two shorter sides. The center part has a substantially        rectangular cross-section with two longer sides and two shorter        sides;    -   two opposing shorter and two opposing longer side walls and    -   a base, wherein said base comprises an opening constructed and        arranged to place the multiwell plate in contact with said        magnetic device and/or a heating device.

In an embodiment of the multiwell plate, adjacent vessels within one roware joined on the longer side of said almost rectangular shape.

For example, the multiwell plate comprises a continuous space which islocated between adjacent rows of vessels. Said continuous space isconstructed and arranged to accommodate a plate-shaped magnetic device.In an embodiment, the bottom part of the vessels comprises a sphericalbottom. In an embodiment, the bottom part of said vessels comprises aconical part located between said central part and said sphericalbottom.

In an embodiment, the top surface comprises ribs, wherein said ribssurround the openings of the vessels. For example, one shorter side ofsaid upper part of the vessels comprises a recess, said recesscomprising a bent surface extending from the rib to the inside of thevessel.

Furthermore, in an embodiment, the vessels comprise a rounded insideshape.

For fixation to the processing or incubation stations, the base maycomprises a rim comprising recesses. Latch clips on a station of ananalyzer can engage with said recesses to fix the plate on a station.

In an embodiment, the vessels comprise an essentially constant wallthickness.

For example, the processing plate (101) in the context of the presentinvention is a 1-component plate. Its top surface (110) comprisesmultiple vessels (103) (FIG. 5, FIG. 6). Each vessel has an opening(108) at the top and is closed at the bottom end (112). The top surface(110) comprises ribs (104) which are may be elevated relative to the topsurface (110) and surround the openings (108) of the vessels (103). Thisprevents contamination of the contents of the vessels (103) withdroplets of liquid that may fall onto the top surface (110) of the plate(101). Views of an exemplary process plate are shown in FIGS. 3 to 8.

The footprint of the processing plate (101) may comprise a length andwidth of the base corresponding to ANSI SBS footprint format. Forexample, the length is 127.76 mm+/−0.25 mm, and the width is 85.48mm+/−0.25 mm. Thus, the plate (101) has two opposing shorter side walls(109) and two opposing longer side walls (118). The processing plate(101) comprises form locking elements (106) for interacting with ahandler (500, FIG. 12). The processing plate (101) can be gripped,transported and positioned quickly and safely at high speed whilemaintaining the correct orientation and position. For example, the formlocking elements (106) for gripping are located within the upper centralpart, for example the upper central third of the processing plate (101).This has the advantage that a potential distortion of the processingplate (101) has only a minor effect on the form locking elements (106)and that the handling of the plate (101) is more robust.

The processing plate (101) may comprise hardware-identifiers (102) and(115). The hardware identifiers (102) and (115) are unique for theprocessing plate (101) and different from hardware identifiers of otherconsumables used in the same system. The hardware identifiers (102, 115)may comprise ridges (119) and/or recesses (125) on the side walls of theconsumables, wherein said pattern of ridges (119) and/or recesses (125)is unique for a specific type of consumable, for example the processingplate (101). This unique pattern is also referred to herein as a unique“surface geometry”. The hardware-identifiers (102, 115) ensure that theuser can only load the processing plate (101) into the appropriatestacker position of an analytical instrument in the proper orientation.On the sides of processing plate (101), guiding elements (116) and (117)are comprised (FIG. 3, FIG. 4). They prevent canting of the processingplate (101). The guiding elements (116, 117) allow the user to load theprocessing plates (101) with guiding elements (116, 117) as a stack intoan analytical instrument which is then transferred vertically within theinstrument in a stacker without canting of the plates.

The center part (120) of the vessels (103) has an almost rectangularcross section (FIG. 6, FIG. 7). They are separated along the longer side(118) of the almost rectangular shape by a common wall (113) (FIG. 3).The row of vessels (103) formed thereby has the advantage that despitethe limited space available, they have a large volume, for example 4 ml.Another advantage is that because of the essentially constant wallthickness, the production is very economical. A further advantage isthat the vessels (103) strengthen each other and, thus, a high stabilityof the shape can be obtained.

Between the rows of vessels (103), a continuous space (121) is located(FIG. 6, FIG. 7). The space (121) can accommodate magnets (202, 203) orheating devices (128) (FIG. 11). These magnets (202, 203) and heatingdevices (128) are for example solid devices. Thus, magnetic particles(216) comprised in liquids (215) which can be held in the vessels (103)can be separated from the liquid (215) by exerting a magnetic field onthe vessels (103) when the magnets (202, 203) are brought into proximityof the vessels (103). Or the contents of the vessels (103) can beincubated at an elevated, controlled temperature when the processingplate (101) is placed on the heating device (128). Since the magnets(202, 203) or heating devices (128) can be solid, a high energy densitycan be achieved. The almost rectangular shape of the central part (120)of the vessels (103) (FIG. 10) also optimizes the contact between thevessel wall (109) and a flat shaped magnet (202) or heating device (128)by optimizing the contact surface between vessel (103) and magnet (202)or heating device (128) and thus enhancing energy transfer into thevessel (103).

In the area of the conical bottom (111) of the vessels, the space (121)is even more pronounced and can accommodate further magnets (203). Thecombination of the large magnets (202) in the upper area and the smallermagnets (203) in the conical area of the vessels allows separation ofmagnetic particles (216) in larger or small volumes of liquid (215). Thesmall magnets (203), thus, make it easier to sequester the magneticparticles (216) during eluate pipetting. This makes it possible topipette the eluate with minimal loss by reducing the dead volume of themagnetic particle (216) pellet. Furthermore, the presence of magneticparticles (216) in the transferred eluate is minimized.

At the upper end of the vessels (103), one of the shorter side walls(109) of the vessel (103) comprises an reagent inlet channel (105) whichextends to the circumferential rib (104) (FIGS. 3, 4, 7). The reagentsare pipetted onto the reagent inlet channel (105) and drain off thechannel (105) into the vessel (103). Thus, contact between the pipetneedle or tip (3, 4) and liquid contained in the vessel is prevented.Furthermore, splashes resulting from liquid being directly dispensedinto another liquid (215) contained in the vessels (103), which maycause contamination of the pipet needle or tip (3, 4) or neighboringvessels (103) is prevented. Sequential pipetting onto the reagent inletchannel (105) of small volumes of reagents followed by the largestvolume of another reagent ensures that the reagents which are only addedin small amounts are drained completely into the vessel (103). Thus,pipetting of small volumes of reagents is possible without loss ofaccuracy of the test to be performed.

On the inside, on the bottom of the vessels (111, 112), the shapebecomes conical (111) and ends with a spherical bottom (112) (FIG. 6.FIG. 7). The inside shape of the vessel (114), including the rectangularcenter part (120), is rounded. The combination of spherical bottom(112), rounded inside shape (114), conical part (111) and refinedsurface of the vessels (103) leads to favorable fluidics whichfacilitate an effective separation and purification of analytes in theprocessing plate (101). The spherical bottom (112) allows an essentiallycomplete use of the separated eluate and a reduction of dead-volumewhich reduces the carryover of reagents or sample cross-contamination.

The rim on the base (129) of the processing plate (101) comprisesrecesses (107) for engagement with latch clips (124) on the processingstation (201) or heating device (128) or analytical instrument (126)(FIG. 5, FIG. 9). The engagement of the latch clips (124) with therecesses (107) allows positioning and fixation of the processing plate(101) on the processing station (201). The presence of the recesses(107) allows the latch force to act on the processing plate (101) almostvertically to the base (129). Thus, only small forces acting sidewayscan occur. This reduces the occurrence of strain, and, thus, thedeformation of the processing plate (101). The vertical latch forces canalso neutralize any deformations of the processing plate (101) leadingto a more precise positioning of the spherical bottoms (111) within theprocessing station (201). In general, the precise interface between theprocessing plate (101) and the processing station (201) or heatingdevice (128) within an analyzer reduces dead-volumes and also reducesthe risk of sample cross-contamination.

A “separation station” is a device or a component of an analyticalsystem allowing for the isolation of the solid support material from theother material present in the fluid sample. Such a separation stationcan e.g. comprise, but is not limited to, a centrifuge, a rack withfilter tubes, a magnet, or other suitable components. In an embodimentof the invention, the separation station comprises one or more magnets.For example, one or more magnets are used for the separation of magneticparticles, for example magnetic glass particles, as a solid support. If,for example, the fluid sample and the solid support material arecombined together in the wells of a multiwell plate, then one or moremagnets comprised by the separation station can e.g. be contacted withthe fluid sample itself by introducing the magnets into the wells, orsaid one or more magnets can be brought close to the outer walls of thewells in order to attract the magnetic particles and subsequentlyseparate them from the surrounding liquid.

In an embodiment, the separation station is a device that comprises amultiwell plate comprising vessels with an opening at the top surface ofthe multiwell plate and a closed bottom. The vessels comprise an upperpart, a center part and a bottom part, wherein the upper part is joinedto the top surface of the multiwell plate and may comprise two longerand two shorter sides. The center part has a substantially rectangularcross-section with two longer sides, wherein said vessels are aligned inrows. A continuous space is located between two adjacent rows forselectively contacting at least one magnet mounted on a fixture with theside walls in at least two Z-positions. The device further comprises amagnetic separation station comprising at least one fixture. The fixturecomprises at least one magnet generating a magnetic field. A movingmechanism is present which vertically moves said at least one fixturecomprising at least one magnet at least between first and secondpositions with respect to the vessels of the multiwell plate. Forexample, said at least two Z-positions of the vessels comprise the sidewalls and the bottom part of said vessels. The magnetic field of said atleast one magnet may draw the magnetic particles to an inner surface ofthe vessel adjacent to said at least one magnet when said at least onemagnet is in said first position. The effect of said magnetic field isless when said at least one magnet is in said second position than whensaid at least one magnet is in said first position. For example, thefixture comprising said at least one magnet comprises a frame. Thevessels have features as described above in the context of multiwellplate/processing plate. One such feature is that at least a part of saidvessels has a substantially rectangular cross-section orthogonal to theaxis of said vessels.

In said first position, said at least one magnet is adjacent to saidpart of said vessels. Adjacent is understood to mean either in closeproximity such as to exert a magnetic field on the contents of thevessel, or in physical contact with the vessel.

The separation station comprises a frame to receive the multiwell plate,and latch-clips to attach the multiwell plate. For example, theseparation station comprises two types of magnets. This embodiment isfurther described below.

A second embodiment is described below, which comprises a spring whichexerts a pressure on the frame comprising the magnets such that themagnets are pressed against the vessels of the multiwell plate.

The first magnets may be constructed and arranged to interact withvessels of a multiwell plate for exerting a magnetic field on a largevolume of liquid comprising magnetic particles held in said vessels.Said second magnets may be constructed and arranged to interact withvessels of a multiwell plate for exerting a magnetic field on a smallvolume of liquid comprising magnetic particles held in said vessels.Said first and second magnets can be moved to different Z-positions.

Useful in the context of the present invention and said separationstation is further a method of isolating and purifying a nucleic acid.The method comprises the steps of binding a nucleic acid to magneticparticles in a vessel of a multiwell plate. The vessel comprises anupper opening, a central part and a bottom part. The bound material isthen separated from unbound material contained in a liquid when themajor part of the liquid is located above the section where the conicalpart of the vessel is replaced by the central part with the rectangularshape, by moving a magnet from a second position to a first positionand, in said first position, applying a magnetic field to the centralpart and, optionally, additionally applying a magnetic field to thebottom part of said vessel. The magnetic particles can optionally bewashed with a washing solution. A small volume of liquid, wherein themajor part of the liquid is located below the section where the conicalpart of the vessel is replaced by the central part with the rectangularshape is separated from said magnetic particles by selectively applyinga magnetic field to the bottom part of said vessel.

Useful in the context of the present invention is also a magneticseparation station for separating a nucleic acid bound to magneticparticles, said separation station comprising first magnets which areconstructed and arranged to interact with vessels of a multiwell platefor exerting a magnetic field on a large volume of liquid comprisingmagnetic particles held in said vessels, and second magnets constructedand arranged to interact with vessels of a multiwell plate for exertinga magnetic field on a small volume of liquid comprising magneticparticles held in said vessels, and wherein said first and secondmagnets can be moved to different Z-positions. Embodiments of themagnetic separation station are described herein.

A first embodiment of a separation station (201) useful for the presentinvention is described below. The first embodiment of said separationstation (201) comprises at least two types of magnets (202, 203). Thefirst, long type of magnet (202) is constructed and arranged to fit intothe space (121) of the processing plate (101). Magnet (202), thus,exerts a magnetic field on the liquid (215) in the vessel (103) tosequester magnetic particles (216) on the inside of the vessel wall.This allows separation of the magnetic particles (216) and any materialbound thereto and the liquid (215) inside the vessel (103) when a largevolume of liquid (215) is present. Magnet (202) has an elongatedstructure and is constructed and arranged to interact with theessentially rectangular central part (120) of the vessel. Thus, magnet(202) is used when the major part of the liquid (215) is located abovethe section where the conical part (111) of the vessel (103) is replacedby the central part (120) with the rectangular shape. As shown in FIG.10, the construction of the magnets (202) comprises fixtures (204, 204a) comprising magnets (202) which fit into the space (121) between therows of vessels (103) in the processing plate (101). Another embodimentof magnets (202) comprises magnets (202) arranged on fixtures (204, 204a). The magnets (203) of the separation station (201) are smaller, andcan interact with the conical part (111) of the vessel (103). This isshown in FIG. 10. Magnets (203) are arranged on a base (205) which canbe moved into the space (121) of the processing plate (101). Each magnet(202, 203) is constructed to interact with two vessels (103) in twoadjacent rows. In an embodiment, the processing plate (101) has 6 rowsof 8 vessels (103). A separation station (201) which can interact withthe processing plate (101) has three fixtures (204, 204 a) comprisingmagnets (202) and four bases (205) comprising magnets (203). Anembodiment is also included wherein the separation station has fourmagnetic fixtures (204, 204 a) comprising magnets (202) and threemagnetic bases (205) comprising magnets (203).

The magnets (202, 203) are movable. The separation station (201)comprises a mechanism to move the fixtures (204, 204 a) and the bases(205). All fixtures (204, 204 a) are interconnected by a base (217) andare, thus, moved coordinately. All magnets (203) are joined to one base(218) and are, thus, moved coordinately. The mechanism for moving themagnetic plates (202) and (203) is constructed and arranged to move thetwo types of magnetic plates (202, 203) to a total of four endpositions:

In FIG. 10 a-c, the magnets (203) are located in proximity of theconical part of the vessels (103) of the processing plate (101). This isthe uppermost position of magnets (203), and is the separation position.In this Figure, the magnets (202) are located in the lowermost position.They are not involved in separation when they are in this position.

In an embodiment shown in FIG. 10, the base (217) of magnets (202) isconnected to a positioning wheel (206). The base (217) comprises abottom end (207) which is flexibly in contact with a connecting element(208) by a moving element (209). Said moving element is constructed andarranged to move the connecting element (208) along a rail (212) fromone side to the other. Said moving element (209) is fixed to theconnecting element (208) with a pin (220). Said connecting element (208)is fixed to the positioning wheel (206) by screw (210). Connectingelement (208) is also connected to axis (211). Said connecting element(208) is a rectangular plate. As the positioning wheel (206) moveseccentrically, around an axis (211), such that the screw (210) movesfrom a point above the eccentric axis to a point below the eccentricaxis, moving element (209) and the bottom end (207) of the base (204)with the magnets (202) attached thereto are moved from the uppermostposition to the lowermost position. The base (218) is mounted on abottom part (219) and is connected, at its lower end, with pin (213) toa moving element (214), which can be a wheel, which interacts with thepositioning wheel (206). When the positioning wheel (206) rotates aroundthe axis (211), wheel (214) moves along positioning wheel (206). If thewheel (214) is located on a section of positioning wheel (206) where thedistance from the axis (211) is short, the magnets (203) are in theirlowermost position. When wheel (214) is located on a section ofpositioning wheel (206) where the distance from the axis (211) is at amaximum, the magnets (203) are in their uppermost position. Thus, in anembodiment of the first embodiment of the separation station, thelocation of the magnets (203) is controlled by the shape of thepositioning wheel (206). When moving element (209) moves along thecentral, rounded upper or lower part (212 a) of rail (212), the smalltype of magnets (203) are moved up and down. When the moving element(209) is located on the side (212 b) of bottom end (207) and moves up ordown, the magnets (202) are moved up- or downwards. The positioningwheel can be rotated by any motor (224).

In an embodiment, a spring (225) is attached to the base (222) of theseparation station and the base (218) of magnets (203) to ensure thatmagnets (203) are moved into the lowermost position when they are moveddownwards.

The term “pin” as used herein relates to any fixation element, includingscrews or pins.

In a second embodiment, the separation station (230) comprises at leastone fixture (231) comprising at least one magnet (232), a number ofmagnets equal to a number of vessels (103) in a row (123). Theseparation station (230) comprises a number of fixtures (231) equal tothe number of rows (123) of the multiwell plate (101) hereinbeforedescribed. For example, six fixtures (231) are mounted on the separationstation (230). At least one magnet (232) is mounted on one fixture(231). For example, the number of magnets (232) equals the number ofvessels (103) in one row (123). For example, eight magnets (232) aremounted on one fixture (231). For example, one type of magnet (232) iscomprised on said fixture (231). For example, the magnet (232) ismounted on one side oriented towards the vessels with which the magnetinteracts.

The fixture (231) is mounted on a base (233). For example, said mount isflexible. The base (233) comprises springs (234) mounted thereon. Thenumber of springs (234) is at least one spring per fixture (231) mountedon said base (233). The base further comprises a chamfer (236) whichlimits the movement of the spring and, consequently, the fixture (231)comprising the magnets (232). For example, any one of said springs (234)is constructed and arranged to interact with a fixture (231). Forexamples, said spring (234) is a yoke spring. Said interaction controlsthe horizontal movement of the fixtures (231). Furthermore, theseparation station (230) comprises a frame (235). The base (233) withfixtures (231) is connected to the frame (235) by a moving mechanism asdescribed hereinbefore for the magnets (232) of the first embodiment.

For example, said base (233) and fixture (231) is constructed andarranged to move vertically (in Z-direction).

The multiwell plate (101) hereinbefore described is inserted into theseparation station (230). The fixture (231) comprising the magnets (232)is moved vertically. Any one fixture (232) is, thus, moved into a space(121) between two rows (123) of vessels (103). The vertical movementbrings the magnets (232) mounted on a fixture (231) into contact withthe vessels (103). The Z-position is chosen depending on the volume ofliquid (215) inside the vessels (103). For large volumes, the magnets(232) contact the vessels (103) in a center position (120) where thevessels (103) are of an almost rectangular shape. For small volumes ofliquid (215) where the major part of the liquid (215) is located belowthe center part (120) of the vessels (103), the magnets (232) contactthe conical part (111) of the vessels (103).

A spring is attached to the base (233) of any one frame (231) (FIG. 9a), b)). The spring presses the magnets (232) against the vessels (103).This ensures a contact between magnets (232) and vessels (103) duringmagnetic separation. For example, the magnet (232) contacts the vessel(103) on the side wall (109) located underneath the inlet (105). Thishas the advantage that liquid which is added by pipetting flows over thesequestered magnetic particles and ensures that particles areresuspended and that all samples in all vessels are treated identically.

This embodiment is particularly suited to separate a liquid (215)comprised in a multiwell plate (101) as hereinbefore described, frommagnetic particles (216) when different levels of liquid (215) arecontained in the vessels (103) of said multiwell plate (101).

A “wash buffer” is a fluid that is designed to remove undesiredcomponents, especially in a purification procedure. Such buffers arewell known in the art. In the context of the purification of nucleicacids, the wash buffer is suited to wash the solid support material inorder to separate the immobilized nucleic acid from any unwantedcomponents. The wash buffer may, for example, contain ethanol and/orchaotropic agents in a buffered solution or solutions with an acidic pHwithout ethanol and/or chaotropic agents as described above. Often thewashing solution or other solutions are provided as stock solutionswhich have to be diluted before use.

The washing in the process according to the invention requires a more orless intensive contact of the solid support material and the nucleicacids immobilized thereon with the wash buffer. Different methods arepossible to achieve this, e.g. shaking the wash buffer with the solidsupport material in or along with the respective vessel or vessels.Another advantageous method is aspirating and dispensing the suspensioncomprising wash buffer and solid support material one or more times.This method may be carried out using a pipet, wherein said pipetcomprises a disposable pipet tip into which said suspension is aspiratedand from which it is dispensed again. Such a pipet tip can be usedseveral times before being discarded and replaced. Disposable pipet tipsuseful for the invention may have a volume of at least 10 μl, or atleast 15 μl, or at least 100 μl, or at least 500 μl, or at least 1 ml,or about 1 ml. Pipets used in the context of the invention can also bepipetting needles.

Thus, an aspect of the invention is the process described above, whereinsaid washing in step d. comprises aspirating and dispensing the washbuffer comprising the solid support material.

For downstream processing of the isolated nucleic acids, it can beadvantageous to separate them from the solid support material beforesubjecting them to amplification.

Therefore, an aspect of the invention is the process described above,wherein said process further comprises in step d. the step of elutingthe purified nucleic acids from the solid support material with anelution buffer after washing said solid support material.

An “elution buffer” in the context of the invention is a suitable liquidfor separating the nucleic acids from the solid support. Such a liquidmay e.g. be distilled water or aqueous salt solutions, such as e.g. Trisbuffers like Tris HCl, or HEPES, or other suitable buffers known to theskilled artisan. The pH value of such an elution buffer can be alkalineor neutral. Said elution buffer may contain further components such ase.g. chelators like EDTA, which stabilizes the isolated nucleic acids byinactivation of degrading enzymes.

The elution can be carried out at elevated temperatures, such that anembodiment of the invention is the process described above, wherein stepd. is carried out at a temperature between 70° C. and 90° C., or at atemperature of 80° C.

“Amplification reagents”, in the context of the invention, are chemicalor biochemical components that enable the amplification of nucleicacids. Such reagents comprise, but are not limited to, nucleic acidpolymerases, buffers, mononucleotides such as nucleoside triphosphates,oligonucleotides e.g. as oligonucleotide primers, salts and theirrespective solutions, detection probes, dyes, and more.

As is known in the art, a “nucleoside” is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are purines andpyrimidines.

“Nucleotides” are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′-, 3′- or 5′-hydroxyl moiety of the sugar. Anucleotide is the monomeric unit of an “oligonucleotide”, which can bemore generally denoted as an “oligomeric compound”, or a“polynucleotide”, more generally denoted as a “polymeric compound”.Another general expression for the aforementioned is deoxyribonucleicacid (DNA) and ribonucleic acid (RNA).

According to the invention, an “oligomeric compound” is a compoundconsisting of “monomeric units” which may be nucleotides alone ornon-natural compounds (see below), more specifically modifiednucleotides (or nucleotide analogs) or non-nucleotide compounds, aloneor combinations thereof.

“Oligonucleotides” and “modified oligonucleotides” (or “oligonucleotideanalogs”) are subgroups of oligomeric compounds. In the context of thisinvention, the term “oligonucleotide” refers to components formed from aplurality of nucleotides as their monomeric units. The phosphate groupsare commonly referred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage. Oligonucleotides and modifiedoligonucleotides (see below) useful for the invention may be synthesizedas principally described in the art and known to the expert in thefield. Methods for preparing oligomeric compounds of specific sequencesare known in the art, and include, for example, cloning and restrictionof appropriate sequences and direct chemical synthesis. Chemicalsynthesis methods may include, for example, the phosphotriester methoddescribed by Narang S. A. et al., Methods in Enzymology 68 (1979) 90-98,the phosphodiester method disclosed by Brown E. L., et al., Methods inEnzymology 68 (1979) 109-151, the phosphoramidite method disclosed inBeaucage et al., Tetrahedron Letters 22 (1981) 1859, the H-phosphonatemethod disclosed in Garegg et al., Chem. Scr. 25 (1985) 280-282 and thesolid support method disclosed in U.S. Pat. No. 4,458,066.

In the process described above, the oligonucleotides may be chemicallymodified, i.e. the primer and/or the probe comprise a modifiednucleotide or a non-nucleotide compound. The probe or the primer is thena modified oligonucleotide.

“Modified nucleotides” (or “nucleotide analogs”) differ from a naturalnucleotide by some modification but still consist of a base, apentofuranosyl sugar, a phosphate portion, base-like, pentofuranosylsugar-like and phosphate-like portion or combinations thereof. Forexample, a label may be attached to the base portion of a nucleotidewhereby a modified nucleotide is obtained. A natural base in anucleotide may also be replaced by e.g. a 7-deazapurine whereby amodified nucleotide is obtained as well.

A “modified oligonucleotide” (or “oligonucleotide analog”), belonging toanother specific subgroup of oligomeric compounds, possesses one or morenucleotides and one or more modified nucleotides as monomeric units.Thus, the term “modified oligonucleotide” (or “oligonucleotide analog”)refers to structures that function in a manner substantially similar tooligonucleotides and can be used interchangeably in the context of thepresent invention. From a synthetical point of view, a modifiedoligonucleotide (or an oligonucleotide analog) can for example be madeby chemical modification of oligonucleotides by appropriate modificationof the phosphate backbone, ribose unit or the nucleotide bases (Uhlmannand Peyman, Chemical Reviews 90 (1990) 543; Verma S., and Eckstein F.,Annu. Rev. Biochem. 67 (1998) 99-134). Representative modificationsinclude phosphorothioate, phosphorodithioate, methyl phosphonate,phosphotriester or phosphoramidate inter-nucleoside linkages in place ofphosphodiester internucleoside linkages; deaza- or azapurines and-pyrimidines in place of natural purine and pyrimidine bases, pyrimidinebases having substituent groups at the 5 or 6 position; purine baseshaving altered substituent groups at the 2, 6 or 8 positions or 7position as 7-deazapurines; bases carrying alkyl-, alkenyl-, alkinyl oraryl-moieties, e.g. lower alkyl groups such as methyl, ethyl, propyl,butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or arylgroups like phenyl, benzyl, naphtyl; sugars having substituent groupsat, for example, their 2′ position; or carbocyclic or acyclic sugaranalogs. Other modifications consistent with the spirit of thisinvention are known to those skilled in the art. Such modifiedoligonucleotides (or oligonucleotide analogs) are best described asbeing functionally interchangeable with, yet structurally differentfrom, natural oligonucleotides. In more detail, exemplary modificationsare disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67(1998) 99-134 or WO 02/12263. In addition, modification can be madewherein nucleoside units are joined via groups that substitute for theinternucleoside phosphate or sugar phosphate linkages. Such linkagesinclude those disclosed in Verma S., and Eckstein F., Annu. Rev.Biochem. 67 (1998) 99-134. When other than phosphate linkages areutilized to link the nucleoside units, such structures have also beendescribed as “oligonucleosides”.

A “nucleic acid” as well as the “target nucleic acid” is a polymericcompound of nucleotides as known to the expert skilled in the art.“Target nucleic acid” is used herein to denote a nucleic acid in asample which should be analyzed, i.e. the presence, non-presence and/oramount thereof in a sample should be determined.

The term “primer” is used herein as known to the expert skilled in theart and refers to oligomeric compounds, primarily to oligonucleotides,but also to modified oligonucleotides that are able to prime DNAsynthesis by a template-dependent DNA polymerase, i.e. the 3′-end of thee.g. primer provides a free 3′-OH group whereto further nucleotides maybe attached by a template-dependent DNA polymerase establishing 3′- to5′-phosphodiester linkage whereby deoxynucleoside triphosphates are usedand whereby pyrophosphate is released.

A “probe” also denotes a natural or modified oligonucleotide. As knownin the art, a probe serves the purpose to detect an analyte oramplificate. In the case of the process described above, probes can beused to detect the amplificates of the target nucleic acids. For thispurpose, probes typically carry labels.

“Labels”, often referred to as “reporter groups”, are generally groupsthat make a nucleic acid, in particular oligonucleotides or modifiedoligonucleotides, as well as any nucleic acids bound theretodistinguishable from the remainder of the sample (nucleic acids havingattached a label can also be termed labeled nucleic acid bindingcompounds, labeled probes or just probes). Exemplary labels according tothe invention are fluorescent labels, which are e.g. fluorescent dyessuch as a fluorescein dye, a rhodamine dye, a cyanine dye, and acoumarin dye. Exemplary fluorescent dyes according to the invention areFAM, HEX, JA270, CAL635, Coumarin343, Quasar705, Cyan500, CY5.5, LC-Red640, LC-Red 705.

In the context of the invention, any primer and/or probe may bechemically modified, i.e. the primer and/or the probe comprise amodified nucleotide or a non-nucleotide compound. The probe or theprimer is then a modified oligonucleotide.

A method of nucleic acid amplification is the Polymerase Chain Reaction(PCR) which is disclosed, among other references, in U.S. Pat. Nos.4,683,202, 4,683,195, 4,800,159, and 4,965,188. PCR typically employstwo or more oligonucleotide primers that bind to a selected nucleic acidtemplate (e.g. DNA or RNA). Primers useful for nucleic acid analysisinclude oligonucleotides capable of acting as a point of initiation ofnucleic acid synthesis within the nucleic acid sequences of the targetnucleic acids. A primer can be purified from a restriction digest byconventional methods, or it can be produced synthetically. The primercan be single-stranded for maximum efficiency in amplification, but theprimer can be double-stranded. Double-stranded primers are firstdenatured, i.e., treated to separate the strands. One method ofdenaturing double stranded nucleic acids is by heating. A “thermostablepolymerase” is a polymerase enzyme that is heat stable, i.e., it is anenzyme that catalyzes the formation of primer extension productscomplementary to a template and does not irreversibly denature whensubjected to the elevated temperatures for the time necessary to effectdenaturation of double-stranded template nucleic acids. Generally, thesynthesis is initiated at the 3′ end of each primer and proceeds in the5′ to 3′ direction along the template strand. Thermostable polymeraseshave e.g. been isolated from Thermus flavus, T. ruber, T. thermophilus,T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, andMethanothermus fervidus. Nonetheless, polymerases that are notthermostable also can be employed in PCR assays provided the enzyme isreplenished.

If the template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 5 sec to 9 min. In order to not expose therespective polymerase like e.g. the Z05 DNA Polymerase to such hightemperatures for too long and thus risking a loss of functional enzyme,it can be preferred to use short denaturation steps.

In an embodiment of the invention, the denaturation step is up to 30sec, or up to 20 sec, or up to 10 sec, or up to 5 sec, or about 5 sec.

If the double-stranded template nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence on the target nucleicacids.

The temperature for annealing can be from about 35° C. to about 70° C.,or about 45° C. to about 65° C.; or about 50° C. to about 60° C., orabout 55° C. to about 58° C. Annealing times can be from about 10 sec toabout 1 min (e.g., about 20 sec to about 50 sec; about 30 sec to about40 sec). In this context, it can be advantageous to use differentannealing temperatures in order to increase the inclusivity of therespective assay. In brief, this means that at relatively low annealingtemperatures, primers may also bind to targets having single mismatches,so variants of certain sequences can also be amplified. This can bedesirable if e.g. a certain organism has known or unknown geneticvariants which should also be detected. On the other hand, relativelyhigh annealing temperatures bear the advantage of providing higherspecificity, since towards higher temperatures the probability of primerbinding to not exactly matching target sequences continuously decreases.In order to benefit from both phenomena, in some embodiments of theinvention the process described above comprises annealing at differenttemperatures, for example first at a lower, then at a highertemperature. If, e.g., a first incubation takes place at 55° C. forabout 5 cycles, non-exactly matching target sequences may be(pre-)amplified. This can be followed e.g. by about 45 cycles at 58° C.,providing for higher specificity throughout the major part of theexperiment. This way, potentially important genetic variants are notmissed, while the specificity remains relatively high.

The reaction mixture is then adjusted to a temperature at which theactivity of the polymerase is promoted or optimized, i.e., a temperaturesufficient for extension to occur from the annealed primer to generateproducts complementary to the nucleic acid to be analyzed. Thetemperature should be sufficient to synthesize an extension product fromeach primer that is annealed to a nucleic acid template, but should notbe so high as to denature an extension product from its complementarytemplate (e.g., the temperature for extension generally ranges fromabout 40° to 80° C. (e.g., about 50° C. to about 70° C.; about 60° C.).Extension times can be from about 10 sec to about 5 min, or about 15 secto 2 min, or about 20 sec to about 1 min, or about 25 sec to about 35sec. The newly synthesized strands form a double-stranded molecule thatcan be used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target nucleic acids. The limiting factors in the reaction arethe amounts of primers, thermostable enzyme, and nucleosidetriphosphates present in the reaction. The cycling steps (i.e.,denaturation, annealing, and extension) can be repeated at least once.For use in detection, the number of cycling steps will depend, e.g., onthe nature of the sample. If the sample is a complex mixture of nucleicacids, more cycling steps will be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, but may be repeated as many as 40, 60,or even 100 times.

Within the scope of the invention, a PCR can be carried out in which thesteps of annealing and extension are performed in the same step(one-step PCR) or, as described above, in separate steps (two-step PCR).Performing annealing and extension together and thus under the samephysical and chemical conditions, with a suitable enzyme such as, forexample, the Z05 DNA polymerase, bears the advantage of saving the timefor an additional step in each cycle, and also abolishing the need foran additional temperature adjustment between annealing and extension.Thus, the one-step PCR reduces the overall complexity of the respectiveassay.

In general, shorter times for the overall amplification can bepreferred, as the time-to-result is reduced and leads to a possibleearlier diagnosis.

Other nucleic acid amplification methods to be used in the context ofthe invention comprise the Ligase Chain Reaction (LCR; Wu D. Y. andWallace R. B., Genomics 4 (1989) 560-69; and Barany F., Proc. Natl.Acad. Sci. USA 88 (1991) 189-193); Polymerase Ligase Chain Reaction(Barany F., PCR Methods and Applic. 1 (1991) 5-16); Gap-LCR (WO90/01069); Repair Chain Reaction (EP 0439182 A2), 3SR (Kwoh D. Y. etal., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli J. C., etal., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; WO 92/08808), andNASBA (U.S. Pat. No. 5,130,238). Further, there are strand displacementamplification (SDA), transcription mediated amplification (TMA), andQb-amplification (for a review see e.g. Whelen A. C. and Persing D. H.,Annu. Rev. Microbiol. 50(1996) 349-373; Abramson R. D. and Myers T. W.,Curr Opin Biotechnol 4 (1993) 41-47).

The internal control nucleic acid used in the present invention mayexhibit the following properties relating to its sequence:

-   -   a melting temperature from 55° C. to 90° C., or from 65° C. to        85° C., or from 70° C. to 80° C., or about 75° C.    -   a length of up to 500 bases or base pairs, or from 50 to 300        bases or base pairs, or from 100 to 200 bases or base pairs, or        about 180 bases or base pairs    -   a GC content from 30% to 70%, or from 40% to 60%, or about 50%.

In the context of the invention, a “sequence” is the primary structureof a nucleic acid, i.e. the specific arrangement of the singlenucleobases of which the respective nucleic acids consists. It has to beunderstood that the term “sequence” does not denote a specific type ofnucleic acid such as RNA or DNA, but applies to both as well as to othertypes of nucleic acids such as e.g. PNA or others. Where nucleobasescorrespond to each other, particularly in the case of uracil (present inRNA) and thymine (present in DNA), these bases can be consideredequivalent between RNA and DNA sequences, as well-known in the pertinentart.

Clinically relevant nucleic acids are often DNA which can be derivede.g. from DNA viruses like e.g. Hepatitis B Virus (HBV), Cytomegalovirus(CMV) and others, or bacteria like e.g. Chlamydia trachomatis (CT),Neisseria gonorrhoeae (NG) and others. In such cases, it can beadvantageous to use an internal control nucleic acid consisting of DNA,in order to reflect the target nucleic acids properties.

Therefore, an aspect of the invention is the method described above,wherein said internal control nucleic acid is DNA.

On the other hand, numerous nucleic acids relevant for clinicaldiagnostics are ribonucleic acids, like e.g. the nucleic acids from RNAviruses such as for example Human Immunodeficiency Virus (HIV),Hepatitis C Virus (HCV), the West Nile Virus (WNV), Human PapillomaVirus (HPV), Japanese Encephalitis Virus (JEV), St. Louis EncephalitisVirus (SLEV) and others. The present invention can be readily applied tosuch nucleic acids. In this case, it can be advantageous to use aninternal control nucleic acid consisting of RNA, in order to reflect thetarget nucleic acids properties. If both RNA and DNA are to be analyzedin the process described supra, the internal control nucleic acid can beRNA, as the internal control nucleic acid mimics the most sensitivetarget of an assay involving multiple targets, and RNA targets usuallyhave to be more closely controlled.

Thus, an aspect of the invention is the method described above, whereinsaid internal control nucleic acid is RNA.

Since RNA is more prone to degradation than DNA due to influences suchas alkaline pH, ribonucleases etc., internal control nucleic acids madeof RNA may be provided as armored particles. Armored particles such asespecially armored RNA are described e.g. in EP910643. In brief, theRNA, which can be produced chemically or heterologously e.g. by bacteriasuch as e.g. E. coli, is at least partially encapsulated in a viral coatprotein. The latter confers resistance of the RNA towards externalinfluences, in particular ribonucleases. It must be understood thatinternal control DNA can also be provided as an armored particle. Botharmored RNA and DNA are useful as internal control nucleic acids in thecontext of the invention. In an embodiment, RNA control nucleic acidsare armored with the MS2 coat protein in E. coli. In a furtherembodiment, DNA control nucleic acids are armored using lambda phageGT11.

Therefore, an aspect of the invention is the method described above,wherein said internal control nucleic acid is an armored nucleic acid.

Typically, in amplification-based nucleic acid diagnostics, RNAtemplates are transcribed into DNA prior to amplification and detection.

Hence, an aspect of the invention is the process described above,wherein said amplification reagents comprise a polymerase with reversetranscriptase activity, said process further comprising between step e.and step f the step of incubating in said reaction vessels said purifiednucleic acids with said one or more amplification reagents for a periodof time and under conditions suitable for transcription of RNA by saidpolymerase with reverse transcriptase activity to occur.

A “polymerase with reverse transcriptase activity” is a nucleic acidpolymerase capable of synthesizing DNA based on an RNA template. It isalso capable of the formation of a double-stranded DNA once the RNA hasbeen reverse transcribed into a single strand cDNA. In an embodiment ofthe invention, the polymerase with reverse transcriptase activity isthermostable.

In an embodiment, the process according to the invention comprisesincubating a sample containing an RNA template with an oligonucleotideprimer sufficiently complementary to said RNA template to hybridize withthe latter, and a thermostable DNA polymerase in the presence of atleast all four natural or modified deoxyribonucleoside triphosphates, inan appropriate buffer comprising a metal ion buffer which, in anembodiment, buffers both the pH and the metal ion concentration. Thisincubation is performed at a temperature sufficient for said primer tohybridize to said RNA template and said DNA polymerase to catalyze thepolymerization of said deoxyribonucleoside triphosphates to form a cDNAsequence complementary to the sequence of said RNA template.

As used herein, the term “cDNA” refers to a complementary DNA moleculesynthesized using a ribonucleic acid strand (RNA) as a template. The RNAmay e.g. be mRNA, tRNA, rRNA, or another form of RNA, such as viral RNA.The cDNA may be single-stranded, double-stranded or may behydrogen-bonded to a complementary RNA molecule as in an RNA/cDNAhybrid.

A primer suitable for annealing to an RNA template may also be suitablefor amplification by PCR. For PCR, a second primer, complementary to thereverse transcribed cDNA strand, provides an initiation site for thesynthesis of an extension product.

In the amplification of an RNA molecule by a DNA polymerase, the firstextension reaction is reverse transcription using an RNA template, and aDNA strand is produced. The second extension reaction, using the DNAtemplate, produces a double-stranded DNA molecule. Thus, synthesis of acomplementary DNA strand from an RNA template by a DNA polymeraseprovides the starting material for amplification.

Thermostable DNA polymerases can be used in a coupled, one-enzymereverse transcription/amplification reaction. The term “homogeneous”, inthis context, refers to a two-step single addition reaction for reversetranscription and amplification of an RNA target. By homogeneous it ismeant that following the reverse transcription (RT) step, there is noneed to open the reaction vessel or otherwise adjust reaction componentsprior to the amplification step. In a non-homogeneous RT/PCR reaction,following reverse transcription and prior to amplification one or moreof the reaction components such as the amplification reagents are e.g.adjusted, added, or diluted, for which the reaction vessel has to beopened, or at least its contents have to be manipulated. Bothhomogeneous and non-homogeneous embodiments are comprised by the scopeof the invention.

Reverse transcription is an important step in an RT/PCR. It is, forexample, known in the art that RNA templates show a tendency towards theformation of secondary structures that may hamper primer binding and/orelongation of the cDNA strand by the respective reverse transcriptase.Thus, relatively high temperatures for an RT reaction are advantageouswith respect to efficiency of the transcription. On the other hand,raising the incubation temperature also implies higher specificity, i.e.the RT primers will not anneal to sequences that exhibit mismatches tothe expected sequence or sequences. Particularly in the case of multipledifferent target RNAs, it can be desirable to also transcribe andsubsequently amplify and detect sequences with single mismatches, e.g.in the case of the possible presence of unknown or rare substrains orsubspecies of organisms in the fluid sample.

In order to benefit from both advantages described above, i.e. thereduction of secondary structures and the reverse transcription oftemplates with mismatches, the RT incubation can be carried out at morethan one different temperature.

Therefore, an aspect of the invention is the process described above,wherein said incubation of the polymerase with reverse transcriptaseactivity is carried out at different temperatures from 30° C. to 75° C.,or from 45° C. to 70° C., or from 55° C. to 65° C.

As a further important aspect of reverse transcription, long RT stepscan damage the DNA templates that may be present in the fluid sample. Ifthe fluid sample contains both RNA and DNA species, it is thus favorableto keep the duration of the RT steps as short as possible, but at thesame time ensuring the synthesis of sufficient amounts of cDNA for thesubsequent amplification and optional detection of amplificates.

Thus, an aspect of the invention is the process described above, whereinthe period of time for incubation of the polymerase with reversetranscriptase activity is up to 30 minutes, 20 minutes, 15 minutes, 12.5minutes, 10 minutes, 5 minutes, or 1 minute.

A further aspect of the invention is the process described above,wherein the polymerase with reverse transcriptase activity andcomprising a mutation is selected from the group consisting of a) a CS5DNA polymerase

-   -   b) a CS6 DNA polymerase    -   c) a Thermotoga maritima DNA polymerase    -   d) a Thermus aquaticus DNA polymerase    -   e) a Thermus thermophilus DNA polymerase    -   f) a Thermus flavus DNA polymerase    -   g) a Thermus filiformis DNA polymerase    -   h) a Thermus sp. sps17 DNA polymerase    -   i) a Thermus sp. Z05 DNA polymerase    -   j) a Thermotoga neapolitana DNA polymerase    -   k) a Termosipho africanus DNA polymerase    -   l) a Thermus caldophilus DNA polymerase

Particularly suitable for these requirements are enzymes carrying amutation in the polymerase domain that enhances their reversetranscription efficiency in terms of a faster extension rate.

Therefore, an aspect of the invention is the process described above,wherein the polymerase with reverse transcriptase activity is apolymerase comprising a mutation conferring an improved nucleic acidextension rate and/or an improved reverse transcriptase activityrelative to the respective wildtype polymerase.

In an embodiment, in the process described above, the polymerase withreverse transcriptase activity is a polymerase comprising a mutationconferring an improved reverse transcriptase activity relative to therespective wildtype polymerase.

Polymerases carrying point mutations that render them particularlyuseful in the context of the invention are disclosed in WO 2008/046612.In particular, polymerases to be used in the context of the presentinvention can be mutated DNA polymerases comprising at least thefollowing motif in the polymerase domain:

T-G-R-L-S-S-X_(b7)-X_(b8)-P-N-L-Q-N; wherein X_(b7) is an amino acidselected from S or T and wherein X_(b8) is an amino acid selected fromG, T, R, K, or L, wherein the polymerase comprises 3′-5′ exonucleaseactivity and has an improved nucleic acid extension rate and/or animproved reverse transcription efficiency relative to the wildtype DNApolymerase, wherein in said wildtype DNA polymerase X_(b8) is an aminoacid selected from D, E or N.

One example is mutants of the thermostable DNA polymerase from Thermusspecies Z05 (described e.g. in U.S. Pat. No. 5,455,170), said variationscomprising mutations in the polymerase domain as compared with therespective wildtype enzyme Z05. An embodiment for the method accordingto the invention is a mutant Z05 DNA polymerase wherein the amino acidat position 580 is selected from the group consisting of G, T, R, K andL.

For reverse transcription using a thermostable polymerase, Mn2+=can besthe divalent cation and is typically included as a salt, for example,manganese chloride (MnCl2), manganese acetate (Mn(OAc)2), or manganesesulfate (MnSO4). If MnCl2 is included in a reaction containing 50 mMTricine buffer, for example, the MnCl2 is generally present at aconcentration of 0.5-7.0 mM; 0.8-1.4 mM is preferred when 200 mM of eachdGTP, dATP, dUTP, and, dCTP are utilized; and 2.5-3.5 mM MnCl2 is mostpreferred. Further, the use of Mg2+ a divalent cation for reversetranscription is also in the context of the present invention.

Since it is in the scope of the invention to reverse-transcribe RNAtarget nucleic acids into cDNA while preserving the DNA target nucleicacids so both cDNA and DNA can be used for subsequent amplification, theinternally controlled process described above is particularly useful forthe simultaneous amplification of target nucleic acids derived from bothorganisms having an RNA or organisms having a DNA genome. This advantageconsiderably increases the spectrum of different organisms, especiallypathogens, that can be analyzed under identical physical conditions.

Therefore, an aspect of the invention is the process described above,wherein the at least two target nucleic acids comprise RNA and DNA.

An “organism”, as used herein, means any living single- or multicellularlife form. In the context of the invention, a virus is an organism.

Especially due to an appropriate temperature optimum, enzymes like Tthpolymerase or, for example, the mutant Z05 DNA polymerase mentionedabove are suited to carry out the subsequent step of amplification ofthe target nucleic acids. Exploiting the same enzyme for both reversetranscription an amplification contributes to the ease of carrying outthe process and facilitates its automation, since the fluid sample doesnot have to be manipulated between the RT and the amplification step.

Therefore, in an embodiment, in the process described above the samepolymerase with reverse transcriptase activity is used for reversetranscription and for the amplification in step f. For example, theenzyme is the mutant Z05 DNA polymerase described supra.

In order not to expose the polymerase or other components of thereaction mixture used in the context of the invention to elevatedtemperatures for times longer than necessary, in an embodiment, stepsabove 90° C. are up to 20 sec, or up to 15 sec, or up to 10 sec, or upto 5 sec or 5 sec long. This also reduces the time-to-result and cutsdown the overall required time of the assay.

In such a homogeneous setup, it can be of considerable advantage to sealthe reaction vessels prior to initiating the RT and the amplification,thereby reducing the risk of contamination. Sealing can be e.g. achievedby applying a foil that can be transparent, a cap, or by oil added tothe reaction vessels and forming a lipophilic phase as a sealing layerat the top of the fluid.

Thus, an aspect of the invention is the process described above, furthercomprising after step e. the step of sealing the at least two reactionvessels.

For the ease of handling and to facilitate automation, the at least tworeaction vessels can be combined in an integral arrangement, so they canbe manipulated together.

Consequently, an aspect of the invention is the process described above,wherein the at least two reaction vessels are combined in the sameintegral arrangement.

Integral arrangements can e.g. be vials or tubes reversibly orirreversibly attached to each other or arranged in a rack. For example,the integral arrangement is a multiwell plate.

The target of the amplification step can be an RNA/DNA hybrid molecule.The target can be a single-stranded or double-stranded nucleic acid.Although the most widely used PCR procedure uses a double-strandedtarget, this is not a necessity. After the first amplification cycle ofa single-stranded DNA target, the reaction mixture contains adouble-stranded DNA molecule consisting of the single-stranded targetand a newly synthesized complementary strand. Similarly, following thefirst amplification cycle of an RNA/cDNA target, the reaction mixturecontains a double-stranded cDNA molecule. At this point, successivecycles of amplification proceed as described above.

Since nucleic acid amplification, especially but not only in the case ofPCR, is very efficient if carried out as a cycling reaction, an aspectof the invention is the process described above, wherein theamplification reaction in step f. consists of multiple cycling steps.

Suitable nucleic acid detection methods are known to the expert in thefield and are described in standard textbooks as Sambrook J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N. Y., 1989 and Ausubel F. et al.: CurrentProtocols in Molecular Biology 1987, J. Wiley and Sons, NY. There may bealso further purification steps before the nucleic acid detection stepis carried out as e.g. a precipitation step. The detection methods mayinclude but are not limited to the binding or intercalating of specificdyes as ethidium bromide which intercalates into the double-stranded DNAand changes its fluorescence thereafter. The purified nucleic acid mayalso be separated by electrophoretic methods optionally after arestriction digest and visualized thereafter. There are also probe-basedassays which exploit the oligonucleotide hybridization to specificsequences and subsequent detection of the hybrid.

The amplified target nucleic acids can be detected during or after theamplification reaction in order to evaluate the result of the analysis.Particularly for detection in real time, it is advantageous to usenucleic acid probes.

Thus, an aspect of the invention is the process described above, whereina cycling step comprises an amplification step and a hybridization step,said hybridization step comprising hybridizing the amplified nucleicacids with probes.

It can be favorable to monitor the amplification reaction in real time,i.e. to detect the target nucleic acids and/or their amplificates duringthe amplification itself.

Therefore, an aspect of the invention is the process described above,wherein the probes are labeled with a donor fluorescent moiety and acorresponding acceptor fluorescent moiety.

The methods set out above can be based on Fluorescence Resonance EnergyTransfer (FRET) between a donor fluorescent moiety and an acceptorfluorescent moiety. A representative donor fluorescent moiety isfluorescein, and representative corresponding acceptor fluorescentmoieties include LC-Red 640, LC-Red 705, Cy5, and Cy5.5. Typically,detection includes exciting the sample at a wavelength absorbed by thedonor fluorescent moiety and visualizing and/or measuring the wavelengthemitted by the corresponding acceptor fluorescent moiety. In the processaccording to the invention, detection can be followed by quantitatingthe FRET. For example, detection is performed after each cycling step.For example, detection is performed in real time. By using commerciallyavailable real-time PCR instrumentation (e.g., LightCycler™ or TaqMan®),PCR amplification and detection of the amplification product can becombined in a single closed cuvette with dramatically reduced cyclingtime. Since detection occurs concurrently with amplification, thereal-time PCR methods obviate the need for manipulation of theamplification product, and diminish the risk of cross-contaminationbetween amplification products. Real-time PCR greatly reducesturn-around time and is an attractive alternative to conventional PCRtechniques in the clinical laboratory.

The following patent applications describe real-time PCR as used in theLightCycler™ technology: WO 97/46707, WO 97/46714 and WO 97/46712. TheLightCycler™ instrument is a rapid thermal cycler combined with amicrovolume fluorometer utilizing high quality optics. This rapidthermocycling technique uses thin glass cuvettes as reaction vessels.Heating and cooling of the reaction chamber are controlled byalternating heated and ambient air. Due to the low mass of air and thehigh ratio of surface area to volume of the cuvettes, very rapidtemperature exchange rates can be achieved within the thermal chamber.

TaqMan® technology utilizes a single-stranded hybridization probelabeled with two fluorescent moieties. When a first fluorescent moietyis excited with light of a suitable wavelength, the absorbed energy istransferred to a second fluorescent moiety according to the principlesof FRET. The second fluorescent moiety is generally a quencher molecule.Typical fluorescent dyes used in this format are for example, amongothers, FAM, HEX, CY5, JA270, Cyan and CY5.5. During the annealing stepof the PCR reaction, the labeled hybridization probe binds to the targetnucleic acid (i.e., the amplification product) and is degraded by the 5′to 3′ exonuclease activity of the Taq or another suitable polymerase asknown by the skilled artisan, such as a mutant Z05 polymerase, duringthe subsequent elongation phase. As a result, the excited fluorescentmoiety and the quencher moiety become spatially separated from oneanother. As a consequence, upon excitation of the first fluorescentmoiety in the absence of the quencher, the fluorescence emission fromthe first fluorescent moiety can be detected.

In both detection formats described above, the intensity of the emittedsignal can be correlated with the number of original target nucleic acidmolecules.

As an alternative to FRET, an amplification product can be detectedusing a double-stranded DNA binding dye such as a fluorescent DNAbinding dye (e.g., SYBRGREEN I® or SYBRGOLD® (Molecular Probes)). Uponinteraction with the double-stranded nucleic acid, such fluorescent DNAbinding dyes emit a fluorescence signal after excitation with light at asuitable wavelength. A double-stranded DNA binding dye such as a nucleicacid intercalating dye also can be used. When double-stranded DNAbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

Molecular beacons in conjunction with FRET can also be used to detectthe presence of an amplification product using the real-time PCR methodsof the invention. Molecular beacon technology uses a hybridization probelabeled with a first fluorescent moiety and a second fluorescent moiety.The second fluorescent moiety is generally a quencher, and thefluorescent labels are typically located at each end of the probe.Molecular beacon technology uses a probe oligonucleotide havingsequences that permit secondary structure formation (e.g. a hairpin). Asa result of secondary structure formation within the probe, bothfluorescent moieties are in spatial proximity when the probe is insolution. After hybridization to the amplification products, thesecondary structure of the probe is disrupted and the fluorescentmoieties become separated from one another such that after excitationwith light of a suitable wavelength, the emission of the firstfluorescent moiety can be detected.

Thus, in a method according to the invention is the method describedabove using FRET, wherein said probes comprise a nucleic acid sequencethat permits secondary structure formation, wherein said secondarystructure formation results in spatial proximity between said first andsecond fluorescent moiety.

Efficient FRET can only take place when the fluorescent moieties are indirect local proximity and when the emission spectrum of the donorfluorescent moiety overlaps with the absorption spectrum of the acceptorfluorescent moiety.

Thus, in an embodiment, said donor and acceptor fluorescent moieties arewithin no more than 5 nucleotides of each other on said probe.

In a further embodiment, said acceptor fluorescent moiety is a quencher.

As described above, in the TaqMan format, during the annealing step ofthe PCR reaction, the labeled hybridization probe binds to the targetnucleic acid (i.e., the amplification product) and is degraded by the5′- to 3′-exonuclease activity of the Taq or another suitable polymeraseas known by the skilled artisan, such as a mutant Z05 polymerase, duringthe subsequent elongation phase.

Thus, in an embodiment, in the process described above, amplificationemploys a polymerase enzyme having 5′- to 3′-exonuclease activity.

It is further advantageous to carefully select the length of theamplicon that is yielded as a result of the process described above.Generally, relatively short amplicons increase the efficiency of theamplification reaction. Thus, an aspect of the invention is the processdescribed above, wherein the amplified fragments comprise up to 450bases, up to 300 bases, up to 200 bases, or up to 150 bases.

The internal control nucleic acid used in the present invention canserve as a “quantitative standard nucleic acid” which is apt to be andused as a reference in order to quantify, i.e. to determine the quantityof the target nucleic acids. For this purpose, one or more quantitativestandard nucleic acids undergo all possible sample preparation stepsalong with the target nucleic acids. Moreover, a quantitative standardnucleic acid is processed throughout the method within the same reactionmixture. It must generate, directly or indirectly, a detectable signalboth in the presence or absence of the target nucleic acid. For thispurpose, the concentration of the quantitative standard nucleic acid hasto be carefully optimized in each test in order not to interfere withsensitivity but in order to generate a detectable signal also e.g. atvery high target concentrations. In terms of the limit of detection(LOD, see below) of the respective assay, the concentration range forthe “quantitative standard nucleic acid” is 20-5000×LOD, 20-1000×LOD, or20-5000×LOD. The final concentration of the quantitative standardnucleic acid in the reaction mixture is dependent on the quantitativemeasuring range accomplished.

“Limit of detection” or “LOD” means the lowest detectable amount orconcentration of a nucleic acid in a sample. A low “LOD” corresponds tohigh sensitivity and vice versa. The “LOD” is usually expressed eitherby means of the unit “cp/ml”, particularly if the nucleic acid is aviral nucleic acid, or as IU/ml. “Cp/ml” means “copies per milliliter”wherein a “copy” is copy of the respective nucleic acid. IU/ml standsfor “International units/ml”, referring to the WHO standard.

A widely used method for calculating an LOD is “Probit Analysis”, whichis a method of analyzing the relationship between a stimulus (dose) andthe quantal (all or nothing) response. In a typical quantal responseexperiment, groups of animals are given different doses of a drug. Thepercent dying at each dose level is recorded. These data may then beanalyzed using Probit Analysis. The Probit Model assumes that thepercent response is related to the log dose as the cumulative normaldistribution. That is, the log doses may be used as variables to readthe percent dying from the cumulative normal. Using the normaldistribution, rather than other probability distributions, influencesthe predicted response rate at the high and low ends of possible doses,but has little influence near the middle.

The Probit Analysis can be applied at distinct “hitrates”. As known inthe art, a “hitrate” is commonly expressed in percent [%] and indicatesthe percentage of positive results at a specific concentration of ananalyte. Thus for example, an LOD can be determined at 95% hitrate,which means that the LOD is calculated for a setting in which 95% of thevalid results are positive.

In an embodiment, the process described above provides an LOD of 1 to100 cp/ml or 0.5 to 50 IU/ml, or 1 to 75 cp/ml or 0.5 to 30 IU/ml, or 1to 25 cp/ml or 1 to 20 IU/ml.

With respect to some examples of possible target nucleic acids fromcertain viruses, the process described above provides the followingLODs:

-   -   HIV: up to 60 cp/ml, up to 50 cp/ml, up to 40 cp/ml, up to 30        cp/ml, up to 20 cp/ml, or up to 15 cp/ml    -   HBV: up to 10 IU/ml, up to 7.5 IU/ml, or up to 5 IU/ml    -   HCV: up to 10 IU/ml, up to 7.5 IU/ml, or up to 5 IU/ml    -   WNV I: up to 20 cp/ml, up to 15 cp/ml, or up to 10 cp/ml    -   WNV II: up to 20 cp/ml, up to 15 cp/ml, up to 10 cp/ml, or up to        5 cp/ml    -   JEV: up to 100 cp/ml, up to 75 cp/ml, up to 50 cp/ml, or up to        30 cp/ml    -   SLEV: up to 100 cp/ml, up to 75 cp/ml, up to 50 cp/ml, up to 25        cp/ml, or up to 10 cp/ml.

An example of how to perform calculation of quantitative results in theTaqMan format based on an internal control nucleic acid serving as aquantitative standard nucleic acid is described in the following: Atiter is calculated from input data of instrument-corrected fluorescencevalues from an entire PCR run. A set of samples containing a targetnucleic acid and an internal control nucleic acid serving as aquantitative standard nucleic acid undergo PCR on a thermocycler using aspecified temperature profile. At selected temperatures and times duringthe PCR profile samples are illuminated by filtered light and thefiltered fluorescence data are collected for each sample for the targetnucleic acid and the internal control nucleic acid. After a PCR run iscomplete, the fluorescence readings are processed to yield one set ofdye concentration data for the internal control nucleic acid and one setof dye concentration data for the target nucleic acid. Each set of dyeconcentration data is processed in the same manner. After severalplausibility checks, the elbow values (CT) are calculated for theinternal control nucleic acid and the target nucleic acid. The elbowvalue is defined as the point where the fluorescence of the targetnucleic acid or the internal control nucleic acid crosses a predefinedthreshold (fluorescence concentration). Titer determination is based onthe assumptions that the target nucleic acid and the internal controlnucleic acid are amplified with the same efficiency and that at thecalculated elbow value equal amounts of amplicon copies of targetnucleic acid and internal control nucleic acid are amplified anddetected. Therefore, the (CTQS−CTtarget) is linear to log (targetconc/QS conc). In this context, QS denotes the internal control nucleicacid serving as a quantitative standard nucleic acid. The titer T canthen be calculated for instance by using a polynomial calibrationformula as in the following equation:

T′=10(a(CTQS−CTtarget)2+b(CTQS−CTtarget)+c)

The polynomial constants and the concentration of the quantitativestandard nucleic acid are known, therefore the only variable in theequation is the difference (CTQS−CTtarget).

Further, in the sense of the invention, the internal control nucleicacid can serve as a “qualitative internal control nucleic acid”. A“qualitative internal control nucleic acid” is particularly useful forconfirming the validity of the test result of a qualitative detectionassay: Even in the case of a negative result, the qualitative internalcontrol must be detected, otherwise the test itself is considered to beinoperative. However, in a qualitative setup, it does not necessarilyhave to be detected in case of a positive result. As a consequence, itsconcentration must be relatively low. It has to be carefully adapted tothe respective assay and its sensitivity. For example, the concentrationrange for the qualitative internal nucleic acid, i.e. the second controlnucleic acid, will comprise a range of 1 copy per reaction to 1000copies per reaction. In relation to the respective assay's limit ofdetection (LOD), its concentration is between the LOD of an assay andthe 25fold value of the LOD, or between the LOD and 10×LOD. Or, it isbetween 2× and 10×LOD. Or, it is between 5× and 10×LOD. Or, it is 5× or10×LOD.

The internal control nucleic acid as used in the present invention isnot restricted to a particular sequence. It can be advantageous to adddifferent internal control nucleic acids to a fluid samples, but to useonly one of them for amplification e.g. by adding only primers for oneof said internal control nucleic acids. In such embodiments, theinternal control nucleic acid to be amplified in a certain experimentcan be chosen by the person skilled in the art, thus increasingflexibility of the analysis to be carried out. In particularlyadvantageous embodiments, said different internal control nucleic acidscan be comprised by a single nucleic acid construct, e.g. a plasmid or adifferent suitable nucleic acid molecule.

Therefore, an aspect of the invention is the process described above,wherein more than one internal control nucleic acid is added in step a.,but only one of said internal control nucleic acids is amplified in stepf.

The results described above may be adulterated and, for example,comprise false-positives, in the case of cross-contamination withnucleic acids from sources other than the fluid sample. In particular,amplificates of former experiments may contribute to such undesiredeffects. One particular method for minimizing the effects ofcross-contamination of nucleic acid amplification is described in U.S.Pat. No. 5,035,996. The method involves the introduction ofunconventional nucleotide bases, such as dUTP, into the amplifiedproduct and exposing carryover products to enzymatic and/orphysicochemical treatment to render the product DNA incapable of servingas a template for subsequent amplifications. Enzymes for such treatmentsare known in the art. For example, uracil-DNA glycosylase, also known asuracil-N-glycosylase or UNG, will remove uracil residues from PCRproducts containing that base. The enzyme treatment results indegradation of the contaminating carryover PCR product and serves to“sterilize” the amplification reaction.

Thus, an aspect of the invention is the process described above, furthercomprising between step d) and step e) the steps of

-   -   treating the fluid sample with an enzyme under conditions in        which products from amplifications of cross-contaminating        nucleic acids from other samples are enzymatically degraded;    -   inactivating said enzyme.

For example, the enzyme is uracil-N-glycosylase.

In a process according to the invention, all steps are automated.“Automated” means that the steps of a process are suitable to be carriedout with an apparatus or machine capable of operating with little or noexternal control or influence by a human being. Only the preparationsteps for the method may have to be done by hand, e.g. storagecontainers have to be filled and put into place, the choice of sampleshas to be performed by a human being and further steps known to theexpert in the field, e.g. the operation of a controlling computer. Theapparatus or machine may e.g. automatically add liquids, mix the samplesor carry out incubation steps at specific temperatures. Typically, sucha machine or apparatus is a robot controlled by a computer which carriesout a program in which the single steps and commands are specified.

A further aspect of the invention is an analytical system (440) forisolating and simultaneously amplifying at least two target nucleicacids that may be present in a fluid sample, said analytical systemcomprising the following modules:

-   -   a separation station (230) comprising a solid support material,        said separation station being constructed and arranged to        separate and purify a target nucleic acid comprised in a fluid        sample    -   an amplification station (405) comprising at least two reaction        vessels, said reaction vessels comprising amplification        reagents, at least a first purified target nucleic acid in at        least a first reaction vessel and at least a second purified        target nucleic acid in at least a second reaction vessel,        wherein the second target nucleic acid is absent from the first        reaction vessel, an internal control nucleic acid and a        polymerase with reverse transcriptase activity, said polymerase        further comprising a mutation conferring an improved nucleic        acid extension rate and/or an improved reverse transcriptase        activity relative to the respective wildtype polymerase.

An “analytical system” is an arrangement of components such asinstruments interacting with each other with the ultimate aim to analyzea given sample.

The advantages of said analytical system are the same as described suprawith respect to the process according to the invention.

The analytical system (440, FIG. 11) of the present invention is asystem (440) comprising a module (401) for isolating and/or purifying ananalyte. Further, the system (440) additionally comprises a module (403)for analyzing said analyte to obtain a detectable signal. The detectablesignal can be detected in the same module (401, 402, 403) or,alternatively, in a separate module. The term “module” as used hereinrelates to any spatially defined location within the analyzer (400). Twomodules (401, 403) can be separated by walls, or can be in openrelationship. Any one module (401, 402, 403) can be either autonomouslycontrolled, or control of the module (401, 402, 403) can be shared withother modules. For example, all modules are controlled centrally.Transfer between modules (401, 402, 403) can be manual, or automated.Thus, a number of different embodiments of automated analyzers (400) areencompassed by the present invention.

The “separation station” is described supra.

An “amplification station” comprises a temperature-controlled incubatorfor incubating the contents of at least two reaction vessels. It furthercomprises a variety of reaction vessels like tubes or plates, in which areaction for the analysis of the sample such as PCR takes place. Theouter limits or walls of such vessels are chemically inert such thatthey do not interfere with the amplification reaction taking placewithin. For the ease of handling and to facilitate automation, the atleast two reaction vessels can be combined in an integral arrangement,so they can be manipulated together.

Consequently, an aspect of the invention is the analytical systemdescribed above, wherein the at least two reaction vessels are combinedin an integral arrangement.

Integral arrangements can e.g. be vials or tubes reversibly orirreversibly attached to each other or arranged in a rack. For example,the integral arrangement is a multiwell plate.

For example, said multiwell plate is held in a holding station. In anembodiment, one handler transports a multiwell vessel from a holdingstation to an air-lock (460), and a second handler transports saidmultiwell plate from said air-lock to said amplification station,wherein both handlers interact with said multiwell plate by aform-locking interaction.

In an embodiment, the analytical system is fully automated.

In one embodiment, at least two reaction vessels combined in an integralarrangement are transported between stations of the system.

In a second embodiment, the purified target nucleic acid is transferredfrom said separation station to said amplification station. For example,a pipettor comprising pipets with attached pipet tips transfers theliquid comprising the purified nucleic acid.

In a third embodiment, the purified nucleic acid is transferred fromsaid separation station to a reaction vessel in an integral arrangementheld in a holding station. For example, said reaction vessel in anintegral arrangement is then transferred from said holding station tosaid amplification station.

The analytical system according to the invention further comprises apipetting unit. Said pipetting unit comprises at least one pipet, ormultiple pipets. In an embodiment, said multiple pipets are combined inone or more integral arrangements, within which the pipets can bemanipulated individually. Pipets used in the context of the inventioncan be pipets comprising pipet tips as described supra. In anotherembodiment, the pipets are pipetting needles.

Alternatively, a reaction vessel or arrangement of reaction vessels usedfor sample preparation in the separation station and containing thefluid comprising the purified target nucleic acids may be transferredfrom the separation station to the amplification station.

For this purpose, the analytical system according to the inventionfurther comprises a transfer unit, said transfer unit comprising arobotic device, said device comprising a handler.

For the reasons set out above in the context of the process according tothe invention, the following are further aspects of the invention:

-   -   The analytical system (440) described above wherein at least one        reaction vessel comprises an RNA target nucleic acid and a DNA        target nucleic acid.    -   The analytical system (440) described above, wherein at least        one reaction vessel comprises an RNA target nucleic acid, and at        least one other reaction vessel comprises a DNA target nucleic        acid.

For example, the analytical system (440) described above furthercomprises one or more elements selected from the group consisting of:

-   -   a detection module (403) for detecting signals evoked by an        analyte    -   a sealer (410)    -   a storage module (1008) for reagents and/or disposables.    -   a control unit (1006) for controlling system components.

A “detection module” (403) can e.g. be an optical detection unit fordetecting the result or the effect of the amplification procedure. Anoptical detection unit may comprise a light source, e.g. a xenon lamp,optics such as mirrors, lenses, optical filters, fiber optics forguiding and filtering the light, one or more reference channels, or aCCD camera or a different camera.

A “sealer” (410) is constructed and arranged to seal any vessels used inconnection with the analytical system according to the invention. Such asealer can, for example, seal tubes with appropriate caps, or multiwellplates with foil, or other suitable sealing materials.

A “storage module” (1008) stores the necessary reagents to bring about achemical or biological reaction important for analysis of the fluidsample. It can also comprise further components useful for the method ofthe invention, e.g. disposables such as pipet tips or vessels to be usedas reaction vessels within the separation station and/or theamplification station.

For example, the analytical system according to the invention furthercomprises a control unit for controlling system components.

Such a “control unit” (1006) may comprise software for ensuring that thedifferent components of said analytical system work and interactcorrectly and with the correct timing, e.g. moving and manipulatingcomponents such as pipets in a coordinated manner. The control unit mayalso comprise a processor running a real-time operating system (RTOS),which is a multi-tasking operating system intended for real-timeapplications. In other words the system processor is capable of managingreal-time constraints, i.e. operational deadlines from event to systemresponse regardless of system load. It controls in real time thatdifferent units within the system operate and respond correctlyaccording to given instructions.

In an embodiment, the present invention relates to an analytical system(440) for processing an analyte, comprising

a. a first position comprising first receptacles (1001) in lineararrangement comprising liquid samples (1010), a processing plate (101)comprising receptacles (103) in n×m arrangement for holding a liquidsample (1011), a first pipetting device (700) comprising at least twopipetting units (702) in linear arrangement, wherein said pipettingunits (702) are coupled to pipette tips (3, 4), and a tip rack (70)comprising pipette tips (3, 4) in an a×(n×m) arrangement;b. a second position comprising a holder (201, 128) for said processingplate (101), a holder (330) for a multiwell plate, a holder (470) forsaid tip rack (70) and a second pipetting device (35), said secondpipetting device (35) comprising pipetting units (702) in an n×marrangement for coupling to pipette tips (3, 4) (FIG. 12). The term“holder” as used herein relates to any arrangement capable of receivinga rack or a processing plate.

The advantages of the analytical system (440) of the present inventionare as described above for the method of the present invention.

For example, the position of said pipetting units (702) of the firstpipetting device (700) are variable. Exemplary embodiments of said firstpipetting device (700) are described hereinafter.

In one embodiment, the tip rack (70) comprises pipette tips (3, 4) in ana×(n×m) arrangement. For example, a first type (4) and a second type (3)of pipette tips are comprised in the tip rack (70). In this embodiment,the first type of pipette tips (4) is arranged in an n×m arrangement,and the second type of pipette tips (3) is arranged in the n×marrangement. In this context, “n” denotes the number of rows and m thenumber of columns, wherein n is 6 and m is 8. For example, the firsttype of pipette tips (4) has a different volume than the second type ofpipette tips (3), or, the volume of the first type of pipette tips (4)is more than 500 ul, and the volume of the second type of pipette tips(3) is less than 500 ul. In this embodiment, a=2. However, embodimentsof the invention with more than two types of pipette tips, and thus a>2are also included in the present invention.

In one aspect, the analytical system (440) of the present inventioncomprises a control unit (1006) for allocating sample types andindividual tests to individual positions of said processing plate (101).For example, said positions are separate cells (401, 402).

In one aspect of the invention, the system additionally comprises atransfer system (480) for transferring said process plate (101) and saidrack (70) between first (402) and second (401) positions. Embodiments ofsaid transfer system (480) are conveyor belts or, one or more handler.

Furthermore, for example said pipette units of said second pipettingdevice (35) are engaged to pipette tips (3, 4) which were used in thefirst position (402).

An embodiment of the system (440) of the present invention additionallycomprises a third station (403) comprising a temperature-controlledincubator for incubating said analyte with reagents necessary to obtaina detectable signal. Further embodiments of this system are describedhereinafter.

More optimal control of the allocation of samples and tests to the n×marrangement is achieved with a first processor (1004) which is comprisedin said first position (402) to which said control unit (1006) transfersinstructions for allocating sample types and individual tests tospecific positions in the n×m arrangement of vessels (103) of theprocess plate (101), and a second processor (1005) which is comprised insaid second position (401) to which said control unit (1006) transfersinstructions for allocating sample types and individual tests tospecific positions in the n×m arrangement of vessels (103) of theprocess plate.

For example, said system additionally comprises a first processorlocated in said first position, and a second processor located in saidsecond position.

For example, said first processor (1004) controls said first pipettingdevice (700) and said second processor (1005) controls said secondpipetting device (35).

All other embodiments and specific descriptions of embodiments of theanalytical system according to the invention are those mentioned for theprocess according to the invention.

DESCRIPTION OF THE FIGURES

FIG. 1:

Schematic depiction of the sample preparation workflow as used in anembodiment of the invention.

Arrows pointing down denote addition of a component or reagent to eachrespective well of the deepwell plate mentioned above, arrows pointingup their respective removal. These actions were performed manually insteps 2, 3, 4, 21 and 22, by the process head of the apparatus in steps10, 14, 16, 18, and 24, and by the reagent head of the apparatus insteps 5, 6, 7, 11, 15 and 19.

It has to be understood that the volumes used can be adjusted flexiblywithin the spirit of the invention, for example at least about up to 30%of the disclosed values. In particular, in the case of step 2, thesample volume can be variable in order to take into account thedifferent types of fluid samples which may require more or less startingmaterial for obtaining proper results, as known by the artisan. Forexample, the range is from about 100 ul to about 850 ul. Or, it is about100 ul, about 500 ul or about 850 ul. For example, the volume in therespective vessels is adjusted to an identical total volume with thediluent in step 3. For example, as in the scheme shown in FIG. 1, thetotal volume adds up to about 850 ul.

FIG. 2a-g

Growth curves of the amplifications of the target nucleic acids derivedfrom HIV, HBV and CT carried out on a LightCycler480 (Roche DiagnosticsGmbH, Mannheim, Del.) as described in Example 1. The “Signal” indicatedon the y-axis is a normalized fluorescent signal. The x-axis shows thenumber of the respective PCR cycle.

The growth curves of HIV and HBV are shown along with the growth curvesof the corresponding internal control nucleic acid. The respectivetarget nucleic acid curves are represented by straight lines, thecontrol nucleic acid curves by dotted lines.

FIG. 2a : Qualitative HIV assay, measured in the channel for detectionof the target probe.

FIG. 2b : Qualitative HIV assay, measured in the channel for detectionof the control probe.

FIG. 2c : Quantitative HIV assay, measured in the channel for detectionof the target probe.

FIG. 2d : Quantitative HIV assay, measured in the channel for detectionof the control probe.

FIG. 2e : Quantitative HBV assay, measured in the channel for detectionof the target probe.

FIG. 2f : Quantitative HBV assay, measured in the channel for detectionof the control probe.

FIG. 2g : CT assay, measured in the channel for detection of the targetprobe.

FIG. 3:

Perspective view of the processing plate.

FIG. 4:

Perspective view of the processing plate from the opposite angle.

FIG. 5:

Top view of the processing plate.

FIG. 6:

Cross-sectional view along the longer side of the processing plate.

FIG. 7:

A partial view of the cross-sectional view.

FIG. 8:

Perspective view of the longer side of the processing plate.

FIG. 9:

a) to d) show different views of the second embodiment of the magneticseparation station.

FIG. 10:

(a) to (c) show a view of the first embodiment of the magneticseparation station holding the Processing plate, with the first type ofmagnets in the uppermost Z-position, and the second type of magnets inthe lowermost Z-position.

FIG. 11:

Schematic drawings of an analyzer comprising different stations, modulesor cells.

FIG. 12:

Shows an analytical system of the present invention.

FIG. 13:

Linearity of the quantitative HBV assay in EDTA plasma according to thedata in Example 2.

FIG. 14:

Linearity of the quantitative HBV assay in serum according to the datain Example 2.

FIG. 15:

Linearity of the quantitative HCV assay in EDTA plasma according to thedata in Example 2.

FIG. 16:

Linearity of the quantitative HCV assay in serum according to the datain Example 2.

FIG. 17:

Linearity of the quantitative HIV assay in EDTA plasma according to thedata in Example 2.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

This example describes a process for isolating and simultaneouslyamplifying at least a first and a second target nucleic acid using asingle generic internal control nucleic acid.

In brief, in the depicted embodiment, realtime PCR is carried outsimultaneously and under identical conditions on a panel of severaldifferent targets comprising bacteria (Chlamydia trachomatis, CT) aswell as a DNA virus (HBV) and an RNA virus (HIV). All samples wereprocessed and analyzed within the same experiment, i.e. on the samedeepwell plate (for sample preparation) or multiwell plate (foramplification and detection), respectively.

The following samples were prepared and subsequently analyzed:

Reagent Manufacturer: HIV-1M Secondary Standard, 50′000 cp/ML Roche HBVSecondary Standard, 400 IU/ml Roche CT (DNA POS CTL pCHL-1) Roche

Suitable standards or other types of targets are available to theskilled artisan.

The instruments listed in the following table were used according to theinstructions of the respective manufacturer:

Instrument Manufacturer Hamilton Star Hamilton Medical AG (Bonaduz, CH)Light Cycler 480 Roche Diagnostics GmbH (Mannheim, DE) Chameleon SealerK biosystems (Essex, UK) Compressor K biosystems (Essex, UK)

For sample preparation the following reagents were used as diluents:

Reagent Manufacturer: PreservCyt Thin Prep K3 EDTA Plasma, PCR neg.Roche

The following dilutions were prepared in advance and stored overnight(plasma dilutions at −60 to −90° C., PreservCyt dilutions at 2-8° C.):

Target Concentration Matrix HBV 50 IU/ml K3 EDTA plasma HIV-1M 100 cp/mlK3 EDTA plasma CT 2.5 fg/ml PreservCyt

Each respective sample (500 ul) and each respective specimen diluent(350 ul) were pipetted manually into a deepwell plate, wherein eachsample was added to three different wells for triplicate analysis. Toeach well containing an HIV or HBV sample, 50 ul of an internal controlnucleic acid were manually added. For the qualitative HIV assay, an RNAserving as a qualitative control was added (100 armoredparticles/sample). For the quantitative HIV assay, an RNA serving as aquantitative standard was added (500 armored particles/sample). For thequantitative HBV assay, a DNA serving as a quantitative standard wasadded (1E4 copies/sample). The sequence of said control nucleic acidswas identical in all cases and selected from the group of SEQ ID NOs45-48.

The respective control nucleic acid was stored in the following buffer:

IC/IQS - Storage Buffer Conc. or pH Tris (mM) 10 EDTA (mM) 0.1 SodiumAzide (w/v, %) 0.05 Poly rA RNA (mg/l) 20 pH 8

Sample preparation was performed on a Hamilton Star (Hamilton, Bonaduz,CH), following the workflow according to the scheme depicted in FIG. 1and using the following reagents:

Protease reagent Conc. or pH Tris (mM) 10 EDTA (mM) 1 Calcium Chloride 5(mM) Calcium Acetate (mM) 5 Esperase (mg/ml) 80 Glycerin (w/v, %) 50 pH5.5

MGP Reagent Conc. or pH MPG Powder (mg/ml) 60 Tris (mM) 30 Methylparaben(w/v, %) 0.1 Sodium Azide (w/v, %) 0.095 pH 8.5

Lysis Reagent Conc. or pH Guanidine Thiocyanate 4 (M) Sodium Citrate(mM) 50 Polydocanol (w/v, %) 5 Dithiotreitol (w/v, %) 2 pH 5.8

Wash buffer Conc. or pH Sodium Citrate (mM) 7.5 Methylparaben (w/v, %)0.1 pH 4.1

Elution buffer Conc. or pH Tris (mM) 30 Methylparaben (w/v, %) 0.2 pH8.5

After the final step, the process head of the Hamilton Star apparatusadded the respective mastermixes (Mmxs) containing amplificationreagents to each well, mixed the fluids containing the isolated nucleicacids with the Mmx and transferred each resulting mixture to acorresponding well of a microwell plate in which the amplification wascarried out.

The following mastermixes (each consisting of the two reagents R1 andR2) were used:

For HIV:

Concentration/ 50 μl-PCR [μM] R1 Reagent Water (PCR grade) Mn(Ac)₂ *4H₂O (pH 6.1 adjusted with Acetic Acid) 3′000 NaN3/Ri, buffered with 10mM Tris at pH 7 [%] 0.018 R2 Reagent DMSO [%] 5.000% NaN3/Ri, bufferedwith 10 mM Tris at pH 7 [%] 0.027% Potassium acetate pH 7.0 110′000Glycerol [%] 3.000% Tricine pH 8.0 50′000 Igepal [%] 0.024% dGTP 337.5dATP 337.5 dCTP 337.5 dUTP 675 Primers/probes selected from SEQ ID NOs1-35 0.1-0.15 SEQ ID NO 42 0.1 SEQ ID NO 43 0.1 SEQ ID NO 44 0.1Uracil-N-Glycosylase 10 (U/reaction) Z05-D Polymerase 40 (U/reactionn)NTQ21-46A - Aptamer 0.222 Water

For HBV:

Concentration/50 μl-PCR R2 Reagent H20 100 % Tricine 7.7 40 mM Tween0.03 % (v/v) Glycerol 5 % (v/v) KOH 25.2 mM KOAc 121.8 mM NTQ21-46A(Aptamer) 0.2625 uM dGTP 0.42 uM dATP 0.42 uM dCTP 0.42 uM dUTP 0.84 uMSEQ ID NO 36 1.2 uM SEQ ID NO 37 0.1 uM SEQ ID NO 38 1.2 uM SEQ ID NO 420.6 uM SEQ ID NO 43 0.6 uM SEQ ID NO 44 0.15 uM Z05D Polymerase 35(U/reaction) Uracil-N-Glycosylase 2 (U/reaction) Sodium Azide 0.027 %(m/v) R1 Reagent H20 100 % MgOAc 2.5 mM MnOAc pH 6.1 2.5 mM Sodium Azide0.018 % (m/v)

For CT:

Concentration/ 50 μl-PCR R1 Reagent Water (PCR grade) Mn(Ac)₂ (pH 6.5 in0.002% (V/V) Glacial 2.7 mM Acetic Acid) NaN3 0.0135% (W/V) R2 ReagentNaN3/Ri, buffered with 10 mM Tris at pH 7 [%] 0.0315% Potassium acetate112.4 mM Glycerol [%]   3.5% Tricine 61 mM Potassium hydroxide 28.4 mMdGTP 525 uM dATP 525 uM dCTP 525 uM dUTP 1.05 mM SEQ ID NO 39 750 nM SEQID NO 40 600 nM SEQ ID NO 41 116 nM Aptamer NTQ-46A 175 nMUracil-N-Glycosylase 5 U/reaction Z05-D Polymerase 31 U/reaction

For amplification and detection, the microwell plate was sealed with anautomated plate sealer (see above), and the plate was transferred to aLightCycler 480 (see above).

The following PCR profile was used:

Thermo Cycling Profile

Program Target Acquisition Hold Ramp Rate Analysis Name (° C.) Mode(hh:mm:ss) (° C./s) Cycles Mode Pre-PCR 50 None 00:02:00 4.4 1 None 94None 00:00:05 4.4 55 None 00:02:00 2.2 60 None 00:06:00 4.4 65 None00:04:00 4.4 1st 95 None 00:00:05 4.4 5 Quantification Measurement 55Single 00:00:30 2.2 2nd 91 None 00:00:05 4.4 45 QuantificationMeasurement 58 Single 00:00:25 2.2 Cooling 40 None 00:02:00 2.2 1 None

Detection Format (Manual)

Filter Combination Integration Time (sec) 435-470 1 495-525 0.5 540-5800.5 610-645 0.5 680-700 1

The Pre-PCR program comprises initial denaturing and incubation at 55,60 and 65° C. for reverse transcription of RNA templates. Incubating atthree temperatures combines the advantageous effects that at lowertemperatures slightly mismatched target sequences (such as geneticvariants of an organism) are also transcribed, while at highertemperatures the formation of RNA secondary structures is suppressed,thus leading to a more efficient transcription.

PCR cycling is divided into two measurements, wherein both measurementsapply a one-step setup (combining annealing and extension). The first 5cycles at 55° C. allow for an increased inclusivity by pre-amplifyingslightly mismatched target sequences, whereas the 45 cycles of thesecond measurement provide for an increased specificity by using anannealing/extension temperature of 58° C.

Using this profile on all samples comprised on the microwell platementioned above, amplification and detection was achieved in allsamples, as depicted in FIG. 2. This shows that the sample preparationprior to amplification was also successfully carried out.

The results for the qualitative and quantitative HIV internal controlsand the quantitative HBV internal control are depicted separately inFIG. 2 for the sake of clarity. It can be seen that the controls werealso successfully amplified in all cases. The quantitation of the HIVand HBV targets in the quantitative setup were calculated by comparisonwith the internal control nucleic acid serving as a quantitativestandard.

Example 2

The generic amplification process described hereinabove was carried outon a variety of different target nucleic acids in separate experimentsbut under identical conditions. Isolation of the respective nucleic acidwas carried out as described under Example 1.

The respective generic internal control nucleic acid was selected fromSEQ ID NOs 45-49 and was armored RNA for RNA targets and lambda-packagedDNA for DNA targets. For qualitative RNA assays, 300 particles wereadded per sample, for quantitative RNA assays 3000 and for all DNAassays 500.

The following PCR profile was used on all targets:

Target Acquisition Plateau Measure Ramp Rate [° C.] Mode [hh:mm:ss][hh:mm:ss] [° C./s] Pre-PCR UNG-Step 50 none 00:02:00 00:00:00 2.2UNG/Template 94 none 00:00:05 00:00:00 4.4 Denaturation RT-Step 55 none00:02:00 00:00:00 2.2 60 none 00:06:00 00:00:00 4.4 65 none 00:04:0000:00:00 4.4 1st 95 none 00:00:05 00:00:00 4.4 Measurement 55 single00:00:30 00:00:08 2.2 2nd 91 none 00:00:05 00:00:00 4.4 Measurement 58single 00:00:25 00:00:08 2.2 Cooling 40 none 00:02:00 00:00:00 2.2 NameCycles Pre-PCR 1 1st 5 Measurement 2nd 45 Measurement Cooling 1

In detail, the following experiments were performed:

1. Qualitative Multiplex Analysis of HBV, HCV and HIV

-   -   a. Mastermix

R1

Conc. in 50 ul-PCR (uM) Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic3′300 Acid) NaN3/Ri, buffered with 10 mM Tris at pH 7 0.018 pH: 6.41

R2:

Conc. in 50 ul-PCR Reagent (uM) DMSO (%) 5.4 NaN3/Ri, buffered with 10mM Tris at pH 7 0.027 KOAc (pH 7.0) 120′000 Glycerol (%) 3 Tween 20 (%)0.015 Tricine pH 8.0 60′000 NTQ21-46A - Aptamer 0.2222Uracil-N-Glycosylase (U/uL) 0.2 dGTP 400.0 dATP 400.0 dCTP 400.0 dUTP800.0 ZO5-D Polymerase (U/ul)* 0.9 Primers/probes selected from SEQ IDNOs 1-35 0.125-0.3  SEQ ID NO 36 0.100 SEQ ID NO 37 0.100 SEQ ID NO 380.150 Primers/probes selected from SEQ ID NOs 60-76 0.050-0.250 SEQ IDNO 42 0.200 SEQ ID NO 43 0.200 SEQ ID NO 44 0.100

Analytical Sensitivity/LOD

For each detected virus (HIV-1 group M, HIV-1 group O, HIV-2, HBV andHCV), at several concentrations/levels at and around the anticipated LODfor EDTA-plasma. One panel per virus and concentration was tested withat least 20 valid replicates per concentration. The LOD was determinedby PROBIT analysis (see Table 1-5).

HIV

TABLE 1 HIV-1 Group M Hit rates and Probit LOD from individual panelNumber of Number of Concentration replicates positives Hit rate 32cp/mL  21 21 100% 16 cp/mL  21 21 100% 8 cp/mL 21 21 100% 4 cp/mL 21 2095% 2 cp/mL 21 15 71% 1 cp/mL 21 9 43% 0 cp/mL (neg. control) 12 0 0%LOD by PROBIT analysis (95% hitrate)   4.06 cp/mL 95% confidenceinterval for LOD by 2.85-9.24 cp/mL PROBIT analysis

Titer of WHO Standard for HIV-1 Group M was converted to IU/mL.

${{Titer}\left( {{in}\frac{IV}{mL}} \right)} = \frac{{Titer}\left( {i\; n\frac{cp}{mL}} \right)}{0.6}$

Therefore HIV-1 Group M LOD in IU/mL is

LOD by PROBIT analysis (95% hitrate): 6.77 IU/mL

95% confidence interval for LOD by PROBIT analysis: 4.75-15.4 IU/mL

TABLE 2 HIV-1 Group O Hit rates and Probit LOD from individual panelNumber of Number of Concentration replicates positives Hit rate 60 cp/mL21 21 100% 30 cp/mL 20 20 100% 20 cp/mL 21 21 100% 14 cp/mL 21 19 90% 7cp/mL 21 15 71% 4.5 cp/mL 21 12 57% 0 cp/mL 12 0 0% (neg. control) LODby PROBIT analysis   14.9 cp/mL (95% hitrate) 95% confidence intervalfor LOD by 10.9-31.5 cp/mL PROBIT analysis

Titer of Primary Standard for HIV-1 Group O was reassigned to CBER HIV-1Group O panel; calculation factor is 0.586.

Therefore HIV-1 Group O LOD is

LOD by PROBIT analysis (95% hitrate): 8.8 cp/mL

95% confidence interval for LOD by PROBIT analysis: 6.4-18.5 cp/mL

TABLE 3 HIV-2 Hit rates and Probit LOD from individual panel Number ofNumber of Concentration replicates positives Hit rate 4 cp/mL 21 21 100%2 cp/mL 21 21 100% 1 cp/mL 21 20 95% 0.5 cp/mL 21 13 62% 0.25 cp/mL 2113 62% 0.125 cp/mL 21 7 33% 0 cp/mL 12 0 0% (neg. control) LOD by PROBITanalysis (95% hitrate)  1.29 cp/mL 95% confidence interval −3.11 cp/mLfor LOD by PROBIT analysis

Titer of Primary Standard for HIV-2 was reassigned to CBER HIV-2 panel;calculation factor is 26.7.

Therefore HIV-2 LOD is

LOD by PROBIT analysis (95% hitrate): 34.44 cp/mL

95% confidence interval for LOD by PROBIT analysis: 21.89-83.04 cp/mL

HBV

TABLE 4 HBV Hit rates and Probit LOD from individual panel Number ofNumber of Concentration replicates positives Hit rate 7.6 IU/mL 21 21100% 3.8 IU/mL 21 21 100% 1.9 IU/mL 21 20 95% 0.95 IU/mL 21 14 67% 0.6IU/mL 19 12 63% 0.4 IU/mL 21 12 57% 0 IU/mL 12 0 0% (neg. control) LODby PROBIT analysis   2.27 IU/mL (95% hitrate) 95% confidence intervalfor 1.48-6.54 IU/mL LOD by PROBIT analysis

HCV

TABLE 5 HCV Hit rates and Probit LOD from individual panel Number ofNumber of Concentration replicates positives Hit rate 24 IU/mL 21 21100% 12 IU/mL 21 21 100% 6 IU/mL 21 21 100% 3 IU/mL 21 17 81% 1.5 IU/mL21 14 67% 0.75 IU/mL 21 9 43% 0 IU/mL 18 0 0% eg. control) LOD by PROBITanalysis    4.76 IU/mL (95% hitrate) 95% confidence interval for3.14-11.61 IU/mL LOD by PROBIT analysis

2. Qualitative Analysis of WNV

Mastermix R1:

Conc. in Reagent 50 ul-PCR (uM) Mn(Ac)2 * 4H2O (pH 6.1 adjusted withAcetic Acid) 3′300 NaN3/Ri, buffered with 10 mM Tris at pH 7 0.018 pH:6.41

R2:

Reagent Conc. in 50 ul-PCR (uM) DMSO (%) 5.4 NaN3/Ri, buffered with 10mM Tris at pH 7 0.027 K acetate pH 7.0 120′000 Glycerol (%) 3 Tween 20(%) 0.015 Tricine pH 8.0 60′000 NTQ21-46A - Aptamer 0.2222Uracil-N-Glycosylase (U/uL) 0.2 dGTP 400.0 dATP 400.0 dCTP 400.0 dUTP800.0 ZO5-D Polymerase (U/ul)* 0.9 Primers/probes selected from SEQ IDNOs 0.08-0.4 53-59 SEQ ID NO 42 0.150 SEQ ID NO 43 0.150 SEQ ID NO 440.100

Analytical Sensitivity/LOD

For the viruses (WNV, SLEV and JEV) an independent panel was prepared asa dilution series of the respective Standard including severalconcentrations/levels at and around the anticipated LOD. One panel pervirus and concentration was tested with at least 20 valid replicates perconcentration. The LOD was determined by PROBIT analysis.

TABLE 6 WNV Hit rates and Probit LOD from individual panel Number ofNumber of Concentration replicates positives Hit rate 20 cp/mL 21 21100% 12 cp/mL 21 21 100% 8 cp/mL 21 21 100% 5 cp/mL 21 17  81% 2.5 cp/mL21 15 71.4%  0.5 cp/mL 21 1  4.8% 0 cp/mL 12 0  0% (neg. control) LOD byPROBIT analysis    6.57 cp/mL (95% hitrate) 95% confidence interval for4.74-11.03 cp/mL LOD by PROBIT analysis

TABLE 7 SLEV Hit rates and Probit LOD from individual panel Number ofNumber of Concentration replicates positives Hit rate 140 cp/mL 21 21 100% 100 cp/mL 21 20 95.2% 70 cp/mL 21 20 95.2% 40 cp/mL 21 17 81.0% 20cp/mL 21 11 52.4% 10 cp/mL 21 6 28.6% 0 cp/mL 12 0   0% (neg. control)LOD by PROBIT analysis    78.9 cp/mL (95% hitrate) 95% confidenceinterval for 55.4-145.7 cp/mL LOD by PROBIT analysis

TABLE 8 JEV Hit rates and Probit LOD from individual panel Number ofNumber of Concentration replicates positives Hit rate 20 cp/mL 21 2095.2% 12 cp/mL 21 20 95.2% 8 cp/mL 21 18 85.7% 5 cp/mL 21 17 81.0% 2.5cp/mL 21 14 66.7% 0.5 cp/mL 21 2 9.52% 0 cp/mL 12 0   0% (neg. control)LOD by PROBIT analysis   13.55 cp/mL (95% hitrate) 95% confidenceinterval for 8.78-27.7 cp/mL LOD by PROBIT analysis

3. Quantitative Analysis of HBV

Mastermix R1:

Final Conc. in 50 Reagent ul-PCR (uM) Mn(Ac)2 * 4H2O (pH 6.1 adjustedwith Acetic Acid) 3′300 NaN3/Ri, buffered with 10 mM Tris at pH 7 0.018pH: 6.41

R2:

Final Conc. in 50 ul-PCR Reagent (uM) Glycerol (%, w/v)    3% Tricine 60mM DMSO (%, v/v)  5.4% KOAc 120 mM Tween 20 (v/v) 0.015% AptamerNTQ21-46 A 0.222 μM ZO5D Polymerase 0.9 U/μL (45 U/rxn)Uracil-N-Glycosylase 0.2 U/μL (10 U/rxn) Sodium Azide (w/v) 0.027% dCTPs400 μM dGTPs 400 μM dATPs 400 μM dUTPs 800 μM SEQ ID NO 36 1.2 μM SEQ IDNO 37 1.2 μM SEQ ID NO 50 0.6 μM SEQ ID NO 51 0.6 μM SEQ ID NO 38 0.1 μMSEQ ID NO 52 M

Analytical Sensitivity/LOD

Four dilution panels were prepared with HBV Secondary Standard(representing Genotype A), i.e., two in HBV negative serum for sampleinput volumes of 200 μL and 500 μL, and two in HBV negative EDTA-plasmafor sample input volumes of 200 μL and 500 μL. Each panel included 7concentration levels at and around the anticipated LOD. One panel permatrix was tested with ≧21 replicates per concentration level. At least20 replicates needed to be valid. The LOD was determined by PROBITanalysis at 95% hit rate and by ≧95% hit rate analysis.

TABLE 9 LOD analysis for 200 μL input volume in EDTA-plasma.* Number ofNumber of Concentration replicates positives Hit rate 25 IU/mL 41 41 100% 15 IU/mL 41 39 95.1% 10 IU/mL 41 40 97.6% 7 IU/mL 41 40 97.6% 4IU/mL 24 20 83.3% 1 IU/mL 24 4 16.7% 0 IU/mL 24 0   0% (neg. control)LOD by PROBIT analysis   8.2 IU/mL (95% hitrate) 95% confidence interval4.8-26.0 IU/mL for LOD by PROBIT analysis *Additional replicates weretested to narrow the observed 95% confidence interval.

TABLE 10 LOD analysis for 500 μL input volume in EDTA-plasma. Number ofNumber of Concentration replicates positives Hit rate 10 IU/mL 21 21100% 7 IU/mL 21 21 100% 4 IU/mL 21 21 100% 2.5 IU/mL 21 20 95.2%  1IU/mL 21 14 66.7%  0.2 IU/mL 21 1  4.8% 0 IU/mL 21 0  0% (neg. control)LOD by PROBIT analysis   2.3 IU/mL (95% hitrate) 95% confidence interval1.6-4.2 IU/mL for LOD by PROBIT analysis

TABLE 11 LOD analysis for 200 μL input volume in serum. Number ofConcentration Number of replicates positives Hit rate 25 IU/mL 21 21 100% 15 IU/mL 21 20 95.2% 10 IU/mL 21 21  100% 7 IU/mL 21 20 95.2% 4IU/mL 21 15 71.4% 1 IU/mL 21 8 38.1% 0 IU/mL 21 0   0% (neg. control)LOD by PROBIT analysis (95% hitrate)   9.4 IU/mL 95% confidence intervalfor LOD by 6.2-19.0 IU/mL PROBIT analysis

TABLE 12 LOD analysis for 500 μL input volume in serum. Number ofConcentration Number of replicates positives Hit rate 10 IU/mL 21 21100% 7 IU/mL 21 21 100% 4 IU/mL 21 21 100% 2.5 IU/mL 21 16 76.2%  1IU/mL 21 16 76.2%  0.2 IU/mL 21 7 33.3%  0 IU/mL 21 0  0% (neg. control)LOD by PROBIT analysis (95% hitrate)   4.1 IU/mL 95% confidence intervalfor LOD by 2.4-10.0 IU/mL PROBIT analysis

Summary LOD:

EDTA-plasma: The PROBIT analysis at 95% hit rate resulted in an LOD of8.2 IU/mL for 200 μL sample input volume and 2.3 IU/mL for 500 μL sampleinput volume for EDTA-plasma.

The 95% confidence interval range for these concentrations was 4.8-26.0IU/mL for 200 μL sample input volume and 1.6-4.2 IU/mL for 500 μL sampleinput volume.

Serum: The PROBIT analysis at 95% hit rate resulted in an LOD of 9.02IU/mL for 200 μL sample input volume and 4.1 IU/mL for 500 μL sampleinput volume for serum.

The 95% confidence interval range for these concentrations was 6.2-19.0IU/mL for 200 μL sample input volume and 2.4-10.0 IU/mL for 500 μLsample input volume.

Linearity

One EDTA-plasma panel and one serum panel were prepared by using HBVgenotype A (provided by RMD Research Pleasanton, linearized plasmid,pHBV-PC_ADW2). Each of the panels was analyzed at 12 concentrationlevels for the determination of the expected dynamic range (4-2E+09IU/mL) of the assay. All concentration levels/panel members (PM) weretested in 21 replicates.

This study was done with a sample input volume of 500 μL. Theconcentration levels were selected as follows: One level below expectedLower Limit of Quantitation (LLOQ), one at expected LLOQ, one aboveexpected LLOQ, several concentrations at intermediates levels, atexpected Upper Limit of Quantitation (ULOQ) and one above expected ULOQ:

PM 12-2.0E+09 IU/mL—above expected ULOQPM 11-1.0E+09 IU/mL—at expected ULOQPM 10-1.0E+08 IU/mL—below expected ULOQPM 9-1.0E+07 IU/mL—intermediate concentration levelPM 8-1.0E+06 IU/mL—intermediate concentration levelPM 7-1.0E+05 IU/mL—intermediate concentration levelPM 6-1.0E+04 IU/mL—intermediate concentration levelPM 5-1.0E+03 IU/mL—intermediate concentration levelPM 6a-2.0E+02 IU/mL—intermediate concentration level (PM 6 diluted to2.0E+02 IU/mL, used for titer assignment of serum panel)PM 4-1.0E+02 IU/mL—intermediate concentration level (also used for titerassignment of plasma panel)PM 3-5.0E+01 IU/mL—above expected LLOQPM 2-1.0E+01 IU/mL—at expected LLOQPM 1-4.0E+00 IU/mL below expected LLOQ

For every valid sample of the linearity panel, the observed HBV DNAtiter was transformed to log 10 titer and the mean log 10 titer wascalculated per concentration level.

TABLE 13 Linearity in EDTA Plasma Nominal Assigned Mean Titer TiterAssigned Log 10 Titer Repli- (IU/mL) (IU/mL) Log10 Titer observed cates4.00E+00 3.50E+00 0.54 0.52 17 1.00E+01 8.70E+00 0.94 0.91 21 5.00E+014.40E+01 1.64 1.69 21 1.00E+02 8.70E+01 1.94 2.04 21 1.00E+03 8.70E+022.94 3.01 21 1.00E+04 8.70E+03 3.94 3.9 21 1.00E+05 8.70E+04 4.94 4.8821 1.00E+06 8.70E+05 5.94 5.87 21 1.00E+07 8.70E+06 6.94 6.92 211.00E+08 8.70E+07 7.94 8.01 21 1.00E+09 8.70E+08 8.94 9.04 21 2.00E+091.70E+09 9.24 9.38 21

A graphical depiction of this result is shown in FIG. 13.

TABLE 14 Linearity in Serum Nominal Assigned Mean Titer Titer AssignedLog 10 Titer Repli- (IU/mL) (IU/mL) Log10 Titer observed cates 4.00E+003.30E+00 0.52 0.7 21 1.00E+01 8.30E+00 0.92 0.99 21 5.00E+01 4.10E+011.62 1.73 21 1.00E+02 8.30E+01 1.92 2.03 21 1.00E+03 8.30E+02 2.92 2.9321 1.00E+04 8.30E+03 3.92 3.8 21 1.00E+05 8.30E+04 4.92 4.78 21 1.00E+068.30E+05 5.92 5.75 21 1.00E+07 8.30E+06 6.92 6.73 21 1.00E+08 8.30E+077.92 7.78 21 1.00E+09 8.30E+08 8.92 8.92 21 2.00E+09 1.70E+09 9.22 9.2221

A graphical depiction of this result is shown in FIG. 14.

Summary Linearity:

The linear range, defined as the concentration range for which the log10 deviation of the mean log 10 observed titers is within ±0.3 of thelog 10 nominal titer was determined as: 3.5E+00 IU/mL-1.7E+09 IU/mL forEDTA-plasma and 3.3E+00 IU/mL-1.7E+09 IU/mL for serum. The Lower Limitof Quantitation was found to be: 4.0E+00 IU/mL for EDTA-plasma andserum.

4. Quantitative Analysis of HCV

Mastermix R1:

Final Conc. in 50 Reagent ul-PCR (uM) Mn(Ac)2 * 4H2O (pH 6.1 adjustedwith Acetic Acid) 3′300 NaN3/Ri, buffered with 10 mM Tris at pH 7 0.018pH: 6.41

R2:

Final Conc. in 50 Reagent ul-PCR Glycerol (%, w/v)   3% Tricine 60 mMDMSO (%, v/v) 5.4% KOAc 120 mM Tween 20 (v/v) 0.015%  NTQ21-46 A 0.222μM ZO5D 0.9 U/μL (45 U/rxn) UNG 0.2 U/μL (10 U/rxn) Sodium Azide (w/v)0.027 dCTPs 400 μM dGTPs 400 μM dATPs 400 μM dUTPs 800 μM Primers/probesselected from SEQ ID NOs 60-76 0.1 μM SEQ ID NO 42 0.3 μM SEQ ID NO 430.3 μM SEQ ID NO 44 μM

Analytical Sensitivity/LOD

A dilution panel was prepared with Roche HCV Secondary Standard in HCVnegative EDTA plasma and serum using a sample input volumes of 200 μLand 500 μL. Each concentration level was tested with 21 replicates. Atleast ≧20 replicates have to be valid. The LOD was determined by PROBITanalysis at 95% hit rate and by ≧95% hit rate analysis.

TABLE 15 Hit rates and Probit with 200 μL sample process input volumefor EDTA-plasma Number of Number of Concentration replicates positivesHit rate 55 IU/mL 21 21 100%  38 IU/mL 21 21 100%  25 IU/mL 21 20 95%12.5 IU/mL   21 19 90%  6 IU/mL 21 15 71%  3 IU/mL 21 6 29% 0 IU/mL(neg. control) 21 0  0% LOD by PROBIT analysis (95% hitrate) 17.4 IU/mL95% confidence interval for LOD by PROBIT 12.1-34.3 analysis IU/mL

TABLE 16 Hit rates and Probit with 500 μL sample process input volumefor EDTA-plasma Number of Number of Concentration replicates positivesHit rate 22 IU/mL 21 21 100%  15 IU/mL 21 21 100%  10 IU/mL 20 20 100%  5 IU/mL 21 19 76% 2.5 IU/mL  21 15 71%  1 IU/mL 21 6 57% 0 IU/mL (neg.control) 21 0  0% LOD by PROBIT analysis (95% hitrate) 9.0 IU/mL 95%confidence interval for LOD by PROBIT 5.5-25.4 analysis IU/mL

TABLE 17 Hit rates and Probit with 200 μL sample process input volumefor serum Number of Number of Concentration replicates positives Hitrate 55 IU/mL 21 21 100%  38 IU/mL 21 21 100%  25 IU/mL 21 20 95% 12.5IU/mL   21 18 86%  6 IU/mL 21 13 62%  3 IU/mL 21 6 29% 0 IU/mL (neg.control) 21 0  0% LOD by PROBIT analysis (95% hitrate) 20.2 IU/mL 95%confidence interval for LOD by PROBIT 14.0-39.3 analysis IU/mL

TABLE 18 Hit rates and Probit with 500 μL sample process input volumefor serum Number of Number of Concentration replicates positives Hitrate 22 IU/mL 21 21 100%  15 IU/mL 21 21 100%  10 IU/mL 21 20 95%  5IU/mL 21 18 86% 2.5 IU/mL  21 12 57%  1 IU/mL 21 4 19% 0 IU/mL (neg.control) 21 0  0% LOD by PROBIT analysis (95% hitrate) 8.2 IU/mL 95%confidence interval for LOD by PROBIT 5.8-15.0 analysis IU/mL

Summary LOD:

1. The PROBIT analysis at 95% Hit rate resulted in an LOD of 17.4 IU/mLfor 200 μL sample process input volume and 9.0 IU/mL for 500 μL sampleprocess input volume for EDTA plasma. The 95% confidence interval forthese concentrations is 12.1-34.3 IU/mL for 200 μL sample process inputvolume and 5.5-25.4 IU/mL for 500 μL sample process input volume.2. The values of the PROBIT analysis at 95% Hit rate is 20.2 IU/mL for200 μL sample process input volume and 8.2 IU/mL for 500 μL sampleprocess input volume for serum. The 95% confidence interval for theseconcentrations is 14.0-39.3 IU/mL for 200 μL sample process input volumeand 5.8-15.0 IU/mL for 500 μL sample process input volume.

Linearity

One preparation of an EDTA-plasma panel and one preparation of a serumpanel of HCV aRNA traceable to the HCV WHO Standard were analyzed. Thelinearity panels were prepared by serial dilution and analyzed at 10different concentrations. The study was done with 500 μL sample processinput volume. The concentrations were selected as follows: One levelbelow expected Lower Limit of Quantification (LLoQ), one at LLoQ, oneabove LLOQ, several concentrations at intermediates levels, at expectedUpper Limit of Quantification (ULoQ) and one at or above ULoQ. For allconcentrations 21 replicates were tested.

PM 1-2.0E+08 IU/mL—above expected ULoQPM 2-1.0E+08 IU/mL—at expected ULoQPM 3-1.0E+07 IU/mL—below expected ULoQPM 4-1.0E+06 IU/mL—intermediate concentration levelPM 5 1.0E+05 IU/mL—intermediate concentration levelPM 6-1.0E+04 IU/mL—intermediate concentration level for titer assignmentPM 7-1.0E+03 IU/mL—intermediate concentration levelPM 8-1.0E+02 IU/mL—above expected LLoQPM 9-1.0E+01 IU/mL—at expected LLoQPM 10-8.0E+00 IU/mL—below expected LLoQ

TABLE 19 Linearity in EDTA Plasma Nominal Assigned Mean Titer TiterAssigned Log 10 Titer Repli- (IU/mL) (IU/mL) Log10 Titer observed cates8.00E+00 4.87E+00 0.7 0.6 15 1.00E+01 6.09E+00 0.8 0.8 17 1.00E+026.09E+01 1.8 1.7 21 1.00E+03 6.09E+02 2.8 2.8 21 1.00E+04 6.09E+03 3.83.8 21 1.00E+05 6.09E+04 4.8 4.7 21/20 1.00E+06 6.09E+05 5.8 5.6 21/201.00E+07 6.09E+06 6.8 6.7 21 1.00E+08 6.09E+07 7.8 7.8/7.7 21/182.00E+08 1.22E+08 8.1 8 21/20

A graphical depiction of this result is shown in FIG. 15.

TABLE 20 Linearity in Serum Nominal Assigned Mean Titer Titer AssignedLog 10 Titer Repli- (IU/mL) (IU/mL) Log10 Titer observed cates 8.00E+003.90E+00 0.6 0.7 10 1.00E+01 4.96E+00 0.7 0.7 14 1.00E+02 4.96E+01 1.71.6 21 1.00E+03 4.96E+02 2.7 2.8 21 1.00E+04 4.96E+03 3.7 3.7 211.00E+05 4.96E+04 4.7 4.7 21 1.00E+06 4.96E+05 5.7 5.7 21 1.00E+074.96E+06 6.7 6.7 21 1.00E+08 4.96E+07 7.7 7.7 21 2.00E+08 9.92E+07 8 8.121

A graphical depiction of this result is shown in FIG. 16.

Summary Linearity:

The linear range, defined as the concentration range for which the log10 deviation of the mean log 10 observed titers is within ±0.3 of thelog 10 nominal titer was determined as: 4.87E+00 IU/mL-1.22E+08 IU/mLfor EDTA-plasma and 3.90E+00 IU/mL-9.92E+07 IU/mL for serum.

5. Quantitative Analysis of HIV

Mastermix R1:

Final Conc. in 50 Reagent ul-PCR (uM) Mn(Ac)2 * 4H2O (pH 6.1 adjustedwith Acetic Acid) 3′300 NaN3/Ri, buffered with 10 mM Tris at pH 7 0.018pH: 6.41

R2:

Final Conc. in 50 Reagent ul-PCR Glycerol (%, w/v)   3% Tricine 60 mMDMSO (%, v/v) 5.4% KOAc 120 mM Tween 20 (v/v) 0.02%  Aptamer NTQ21-46 A0.222 μM ZO5D Polymerase 0.9 U/μL (45 U/rxn) UNG 0.2 U/μL (10 U/rxn)Sodium Azide (w/v) 0.027 dCTPs 400 μM dGTPs 400 μM dATPs 400 μM dUTPs800 μM Primers/probes selected from SEQ ID NOs 1-35 0.1 μM-0.3 μM SEQ IDNO 50 0.3 μM SEQ ID NO 51 0.3 μM SEQ ID NO 52 μM

Analytical Sensitivity/LOD

A dilution panel was prepared with HIV-1M Secondary Standard in HIV-1negative EDTA plasma for sample input volumes of 200 μL and 500 μL. Eachconcentration level was tested with 21 replicates. At least ≧20replicates have to be valid. The LOD was determined by PROBIT analysisat 95% hit rate and by ≧95% hit rate analysis.

TABLE 21 LOD analysis for 200 μL input volume in EDTA-plasma Number ofNumber of Concentration replicates positives Hit rate 200 cp/mL  21 21 100% 100 cp/mL  21 21  100% 80 cp/mL 21 21  100% 50 cp/mL 21 20 95.2%30 cp/mL 21 18 85.7% 20 cp/mL 21 17 81.0% 10 cp/mL 21 8 38.1% 0 cp/mL(neg. control) 21 0   0% LOD by PROBIT analysis (95% hitrate) 41.8 cp/mL95% confidence interval for LOD by PROBIT 30.9-74.9 analysis cp/mL

TABLE 22 LOD analysis for 500 μL input volume Number of Number ofConcentration replicates positives Hit rate 30 cp/mL 21 21  100% 25cp/mL 21 20 95.2% 20 cp/mL 21 21  100% 13.5 cp/mL    21 18 85.7%  9cp/mL 21 13 61.9%  6 cp/mL 21 9 42.9% 0 cp/mL (neg. control) 21 0   0%LOD by PROBIT analysis (95% hitrate) 18.9 cp/mL 95% confidence intervalfor LOD by PROBIT 14.9-29.4 analysis cp/mL

Summary LOD

1. The PROBIT analysis at 95% hit rate resulted in an LOD of 41.8 cp/mLfor 200 μL input volume and 18.9 cp/mL for 500 μL input volume.2. The 95% confidence interval range for these concentrations was30.9-74.9 cp/mL for 200 μL input volume and 14.9-29.4 cp/mL for 500 μLinput volume.

Linearity

The samples used in the Linearity/Dynamic Range/Accuracy study consistedof a dilution panel of an HIV-1 cell culture supernatant material, HIV-1group M subtype B.

The linearity panel was prepared by serial dilution. This panel wasanalyzed at 10 concentration levels.

The concentrations were selected as follows: One level below expectedLower Limit of Quantitation (LLoQ), one at LLoQ, one above LLoQ, severalconcentrations at intermediate levels, at expected Upper Limit ofQuantitation (ULoQ) and one above ULoQ. For all concentrations 21replicates were tested. The linearity study was done with 500 μL inputvolume):

PM 1-2.0E+07 cp/mL—above expected ULoQPM 2-1.0E+07 cp/mL—at expected ULoQPM 3-1.0E+06 cp/mL—below expected ULoQPM 4-1.0E+05 cp/mL—intermediate concentration levelPM 5 3.0E+04 cp/mL—intermediate concentration level for titer assignmentPM 6-1.0E+04 cp/mL—intermediate concentration levelPM 7-1.0E+03 cp/mL—intermediate concentration levelPM 8-1.0E+02 cp/mL—intermediate concentration levelPM 9-5.0E+01 cp/mL—above expected LLoQPM 10-2.0E+01 cp/mL—at expected LLoQPM 11-1.5E+01 cp/mL below expected LLoQ

TABLE 23 Linearity in EDTA Plasma Nominal Assigned Mean Titer TiterAssigned Log 10 Titer Repli- (cp/mL) (cp/mL) Log10 Titer observed cates1.50E+01 1.50E+01 1.2 1.3 21 2.00E+01 2.00E+01 1.3 1.5 21 5.00E+015.10E+01 1.7 1.8 21 1.00E+02 1.00E+02 2 2 21 1.00E+03 1.00E+03 3 3 211.00E+04 1.00E+04 4 4 21 1.00E+05 1.00E+05 5 5 21 1.00E+06 1.00E+06 6 621 1.00E+07 1.00E+07 7 7 21 2.00E+07 2.00E+07 7.3 7.4 21

A graphical depiction of this result is shown in FIG. 17.

Summary Linearity

The linear range, defined as the concentration range for which the log10 deviation of the mean log 10 observed titers is within ±0.3 of thelog 10 nominal titer was determined as 1.5E+01 cp/mL-2.0E+07 cp/mL

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, sequence accessionnumbers, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A process for isolating and simultaneouslyamplifying at least a first and a second target nucleic acid that may bepresent in one or more samples, the process comprising the steps of: a)adding an internal control nucleic acid to each of the one or moresamples; b) combining a solid support material and the one or moresamples in one or more vessels for a period of time, under conditionssufficient to permit nucleic acids comprising the target nucleic acidsand the internal control nucleic acid to be immobilized on the solidsupport material; c) isolating the solid support material from the othermaterial present in the samples in a separation station; d) purifyingthe target nucleic acids in the separation station and washing the solidsupport material one or more times with a wash buffer; e) contacting thepurified target nucleic acids and the purified internal control nucleicacid with one or more amplification reagents, comprising at least onedistinct set of primers for each of the target nucleic acids and for theinternal control nucleic acid in at least two reaction vessels, whereinat least a first reaction vessel comprises at least the first targetnucleic acid, and at least a second reaction vessel comprises at leastthe second target nucleic acid, and wherein the second target nucleicacid is absent from the first reaction vessel; f) incubating in thereaction vessels the purified target nucleic acids and the purifiedinternal control nucleic acid with the one or more amplificationreagents for a period of time, under conditions sufficient for anamplification reaction indicative of the presence or absence of thetarget nucleic acids to occur; and g) detecting and measuring signalsgenerated by the amplification products of the target nucleic acids andbeing proportional to the concentration of the target nucleic acids, anddetecting and measuring a signal generated by the internal controlnucleic acid; wherein the conditions for amplification and detection insteps d) to g) are identical for the at least first and second purifiedtarget nucleic acids and the internal control nucleic acid, and whereinthe sequence of the internal control nucleic acid is identical for theat least first and second purified target nucleic acids.
 2. The processaccording to claim 1, wherein the presence of an amplification productof the internal control nucleic acid is indicative of an amplificationoccurring in the reaction mixture even in the absence of amplificationproducts for one or more of the target nucleic acids.
 3. The processaccording to claim 1, further comprising the following step: h)determining the quantity of one or more of the target nucleic acids. 4.The process according to claim 1, wherein the amplification reagentscomprise a polymerase with reverse transcriptase activity, the processfurther comprising between step e) and step f) the step of incubating inthe reaction vessels the purified nucleic acids with the one or moreamplification reagents for a period of time and under conditionssuitable for transcription of RNA by the polymerase with reversetranscriptase activity to occur.
 5. The process according to claim 1,wherein the internal control nucleic acid is DNA.
 6. The processaccording to claim 1, wherein the internal control nucleic acid is RNA.7. The process according to claim 1, wherein the internal controlnucleic acid is an armored nucleic acid.
 8. The process according toclaim 1, wherein the sequence of the internal control nucleic acid isdifferent from the sequences of the other nucleic acids present in theone or more samples.
 9. The process according to claim 1, wherein thesequence of the internal control nucleic acid is derived from anaturally occurring genome.
 10. The process according to claim 1,wherein the sequence of the internal control nucleic acid is scrambled.11. The process according to claim 1, wherein the internal controlnucleic acid has a melting temperature from 50° C. to 90° C.
 12. Theprocess according to claim 1, wherein the internal control nucleic acidhas a length of up to 500 bases.
 13. The process according to claim 1,wherein the sequence of the internal control nucleic acid has a GCcontent of 30% to 70%.
 14. The process according to claim 1, wherein theinternal control nucleic acid has a concentration between LOD and20×LOD.
 15. The process according to claim 1, wherein the internalcontrol nucleic acid has a concentration between 20×LOD and 5000λLOD.16. The process according to claim 1, wherein more than one internalcontrol nucleic acid is added in step a), but only one of the internalcontrol nucleic acids is amplified in step f).